Search results for the GEO ID: GSE43777  |
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GSM ID | GPL ID |
Select for analysis |
Title |
Source name |
Description |
Characteristics |
GSM1070871 | GPL201 |
|
VFP-0003_G2_DF [HG-Focus]
|
VFP_G2_DF
|
patient no.: 3
Stage: G2
infecting serotype: 1
phase: Early Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 2
scan date: 2008-01-24
|
|
| Sample_geo_accession | GSM1070871
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070871/suppl/GSM1070871_VFP-0003-1_2_.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070872 | GPL201 |
|
VFP-0003_G3_DF [HG-Focus]
|
VFP_G3_DF
|
patient no.: 3
Stage: G3
infecting serotype: 1
phase: Early Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df0
scan date: 2008-01-24
|
|
| Sample_geo_accession | GSM1070872
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070872/suppl/GSM1070872_VFP-0003-5.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070873 | GPL201 |
|
VFP-0008_G5_DF [HG-Focus]
|
VFP_G5_DF
|
patient no.: 8
Stage: G5
infecting serotype: 1
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 5
scan date: 2008-01-24
|
|
| Sample_geo_accession | GSM1070873
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070873/suppl/GSM1070873_VFP-0008-2.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070874 | GPL201 |
|
VFP-0008_G6_DF [HG-Focus]
|
VFP_G6_DF
|
patient no.: 8
Stage: G6
infecting serotype: 1
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df0
scan date: 2008-01-24
|
|
| Sample_geo_accession | GSM1070874
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070874/suppl/GSM1070874_VFP-0008-5.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070875 | GPL201 |
|
VFP-0008_G7_DF [HG-Focus]
|
VFP_G7_DF
|
patient no.: 8
Stage: G7
infecting serotype: 1
phase: Convalenscent
severity: DF (dengue fever)
fever days or defervescent (df) days: c
scan date: 2008-01-24
|
|
| Sample_geo_accession | GSM1070875
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070875/suppl/GSM1070875_VFP-0008-6.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070876 | GPL201 |
|
VFP-0011_G4_DHF [HG-Focus]
|
VFP_G4_DHF
|
patient no.: 11
Stage: G4
infecting serotype: 3
phase: Late Acute
severity: DHF (dengue hemorrhagic fever)
fever days or defervescent (df) days: 4
scan date: 2007-11-07
|
|
| Sample_geo_accession | GSM1070876
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070876/suppl/GSM1070876_VFP-0011-1.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070877 | GPL201 |
|
VFP-0011_G6_DHF [HG-Focus]
|
VFP_G6_DHF
|
patient no.: 11
Stage: G6
infecting serotype: 3
phase: Late Acute
severity: DHF (dengue hemorrhagic fever)
fever days or defervescent (df) days: df1
scan date: 2007-11-07
|
|
| Sample_geo_accession | GSM1070877
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070877/suppl/GSM1070877_VFP-0011-5.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070878 | GPL201 |
|
VFP-0011_G7_DHF [HG-Focus]
|
VFP_G7_DHF
|
patient no.: 11
Stage: G7
infecting serotype: 3
phase: Convalenscent
severity: DHF (dengue hemorrhagic fever)
fever days or defervescent (df) days: c
scan date: 2007-11-07
|
|
| Sample_geo_accession | GSM1070878
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070878/suppl/GSM1070878_VFP-0011-6.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070879 | GPL201 |
|
VFP-0012_G0_DF [HG-Focus]
|
VFP_G0_DF
|
patient no.: 12
Stage: G0
infecting serotype: 3
phase: Early Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 0
scan date: 2007-11-15
|
|
| Sample_geo_accession | GSM1070879
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070879/suppl/GSM1070879_VFP-0012-1.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070880 | GPL201 |
|
VFP-0012_G1_DF [HG-Focus]
|
VFP_G1_DF
|
patient no.: 12
Stage: G1
infecting serotype: 3
phase: Early Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 1
scan date: 2007-11-15
|
|
| Sample_geo_accession | GSM1070880
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070880/suppl/GSM1070880_VFP-0012-2.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070881 | GPL201 |
|
VFP-0012_G2_DF [HG-Focus]
|
VFP_G2_DF
|
patient no.: 12
Stage: G2
infecting serotype: 3
phase: Early Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df0
scan date: 2007-11-15
|
|
| Sample_geo_accession | GSM1070881
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070881/suppl/GSM1070881_VFP-0012-3.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070882 | GPL201 |
|
VFP-0012_G3_DF [HG-Focus]
|
VFP_G3_DF
|
patient no.: 12
Stage: G3
infecting serotype: 3
phase: Early Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df2
scan date: 2007-11-15
|
|
| Sample_geo_accession | GSM1070882
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070882/suppl/GSM1070882_VFP-0012-4.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070883 | GPL201 |
|
VFP-0012_G5_DF [HG-Focus]
|
VFP_G5_DF
|
patient no.: 12
Stage: G5
infecting serotype: 3
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df3
scan date: 2007-11-15
|
|
| Sample_geo_accession | GSM1070883
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070883/suppl/GSM1070883_VFP-0012-5.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070884 | GPL201 |
|
VFP-0012_G7_DF [HG-Focus]
|
VFP_G7_DF
|
patient no.: 12
Stage: G7
infecting serotype: 3
phase: Convalenscent
severity: DF (dengue fever)
fever days or defervescent (df) days: c
scan date: 2007-11-15
|
|
| Sample_geo_accession | GSM1070884
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070884/suppl/GSM1070884_VFP-0012-6.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070885 | GPL201 |
|
VFP-0013_G2_DF [HG-Focus]
|
VFP_G2_DF
|
patient no.: 13
Stage: G2
infecting serotype: 1
phase: Early Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 2
scan date: 2007-11-21
|
|
| Sample_geo_accession | GSM1070885
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070885/suppl/GSM1070885_VFP-0013-1.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070886 | GPL201 |
|
VFP-0013_G3_DF [HG-Focus]
|
VFP_G3_DF
|
patient no.: 13
Stage: G3
infecting serotype: 1
phase: Early Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 3
scan date: 2007-11-21
|
|
| Sample_geo_accession | GSM1070886
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070886/suppl/GSM1070886_VFP-0013-2.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070887 | GPL201 |
|
VFP-0013_G4_DF [HG-Focus]
|
VFP_G4_DF
|
patient no.: 13
Stage: G4
infecting serotype: 1
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 4
scan date: 2007-11-21
|
|
| Sample_geo_accession | GSM1070887
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070887/suppl/GSM1070887_VFP-0013-3.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070888 | GPL201 |
|
VFP-0013_G5_DF [HG-Focus]
|
VFP_G5_DF
|
patient no.: 13
Stage: G5
infecting serotype: 1
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 5
scan date: 2007-11-21
|
|
| Sample_geo_accession | GSM1070888
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070888/suppl/GSM1070888_VFP-0013-4.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070889 | GPL201 |
|
VFP-0013_G6_DF [HG-Focus]
|
VFP_G6_DF
|
patient no.: 13
Stage: G6
infecting serotype: 1
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df0
scan date: 2007-11-21
|
|
| Sample_geo_accession | GSM1070889
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070889/suppl/GSM1070889_VFP-0013-5.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070890 | GPL201 |
|
VFP-0013_G7_DF [HG-Focus]
|
VFP_G7_DF
|
patient no.: 13
Stage: G7
infecting serotype: 1
phase: Convalenscent
severity: DF (dengue fever)
fever days or defervescent (df) days: c
scan date: 2007-11-21
|
|
| Sample_geo_accession | GSM1070890
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070890/suppl/GSM1070890_VFP-0013-6.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070891 | GPL201 |
|
VFP-0014_G4_DF [HG-Focus]
|
VFP_G4_DF
|
patient no.: 14
Stage: G4
infecting serotype: 1
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 4
scan date: 2007-11-07
|
|
| Sample_geo_accession | GSM1070891
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070891/suppl/GSM1070891_VFP-0014-1.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070892 | GPL201 |
|
VFP-0014_G5_DF [HG-Focus]
|
VFP_G5_DF
|
patient no.: 14
Stage: G5
infecting serotype: 1
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df0
scan date: 2007-11-07
|
|
| Sample_geo_accession | GSM1070892
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070892/suppl/GSM1070892_VFP-0014-5.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070893 | GPL201 |
|
VFP-0014_G7_DF [HG-Focus]
|
VFP_G7_DF
|
patient no.: 14
Stage: G7
infecting serotype: 1
phase: Convalenscent
severity: DF (dengue fever)
fever days or defervescent (df) days: c
scan date: 2007-11-07
|
|
| Sample_geo_accession | GSM1070893
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070893/suppl/GSM1070893_VFP-0014-6.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070894 | GPL201 |
|
VFP-0015_G4_DF [HG-Focus]
|
VFP_G4_DF
|
patient no.: 15
Stage: G4
infecting serotype: 1
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 4
scan date: 2007-11-07
|
|
| Sample_geo_accession | GSM1070894
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070894/suppl/GSM1070894_VFP-0015-1.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070895 | GPL201 |
|
VFP-0015_G6_DF [HG-Focus]
|
VFP_G6_DF
|
patient no.: 15
Stage: G6
infecting serotype: 1
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df0
scan date: 2007-11-07
|
|
| Sample_geo_accession | GSM1070895
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070895/suppl/GSM1070895_VFP-0015-5.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070896 | GPL201 |
|
VFP-0015_G7_DF [HG-Focus]
|
VFP_G7_DF
|
patient no.: 15
Stage: G7
infecting serotype: 1
phase: Convalenscent
severity: DF (dengue fever)
fever days or defervescent (df) days: c
scan date: 2007-11-07
|
|
| Sample_geo_accession | GSM1070896
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070896/suppl/GSM1070896_VFP-0015-6.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070897 | GPL201 |
|
VFP-0017_G1_DF [HG-Focus]
|
VFP_G1_DF
|
patient no.: 17
Stage: G1
infecting serotype: 1
phase: Early Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 1
scan date: 2007-11-07
|
|
| Sample_geo_accession | GSM1070897
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070897/suppl/GSM1070897_VFP-0017-1.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070898 | GPL201 |
|
VFP-0017_G3_DF [HG-Focus]
|
VFP_G3_DF
|
patient no.: 17
Stage: G3
infecting serotype: 1
phase: Early Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df1
scan date: 2007-11-07
|
|
| Sample_geo_accession | GSM1070898
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070898/suppl/GSM1070898_VFP-0017-5.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070899 | GPL201 |
|
VFP-0017_G7_DF [HG-Focus]
|
VFP_G7_DF
|
patient no.: 17
Stage: G7
infecting serotype: 1
phase: Convalenscent
severity: DF (dengue fever)
fever days or defervescent (df) days: c
scan date: 2007-11-07
|
|
| Sample_geo_accession | GSM1070899
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070899/suppl/GSM1070899_VFP-0017-6.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070900 | GPL201 |
|
VFP-0020_G4_DF [HG-Focus]
|
VFP_G4_DF
|
patient no.: 20
Stage: G4
infecting serotype: 1
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 4
scan date: 2007-11-09
|
|
| Sample_geo_accession | GSM1070900
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070900/suppl/GSM1070900_VFP-0020-1.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070901 | GPL201 |
|
VFP-0020_G5_DF [HG-Focus]
|
VFP_G5_DF
|
patient no.: 20
Stage: G5
infecting serotype: 1
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df0
scan date: 2007-11-21
|
|
| Sample_geo_accession | GSM1070901
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070901/suppl/GSM1070901_VFP-0020-5.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070902 | GPL201 |
|
VFP-0020_G6_DF [HG-Focus]
|
VFP_G6_DF
|
patient no.: 20
Stage: G6
infecting serotype: 1
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df1
scan date: 2007-11-09
|
|
| Sample_geo_accession | GSM1070902
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070902/suppl/GSM1070902_VFP-0020-6.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070903 | GPL201 |
|
VFP-0021_G2_DF [HG-Focus]
|
VFP_G2_DF
|
patient no.: 21
Stage: G2
infecting serotype: 1
phase: Early Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 2
scan date: 2008-01-25
|
|
| Sample_geo_accession | GSM1070903
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070903/suppl/GSM1070903_VFP-0021-1_2_.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070904 | GPL201 |
|
VFP-0021_G6_DF [HG-Focus]
|
VFP_G6_DF
|
patient no.: 21
Stage: G6
infecting serotype: 1
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df1
scan date: 2008-01-25
|
|
| Sample_geo_accession | GSM1070904
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070904/suppl/GSM1070904_VFP-0021-5.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070905 | GPL201 |
|
VFP-0021_G7_DF [HG-Focus]
|
VFP_G7_DF
|
patient no.: 21
Stage: G7
infecting serotype: 1
phase: Convalenscent
severity: DF (dengue fever)
fever days or defervescent (df) days: c
scan date: 2008-01-25
|
|
| Sample_geo_accession | GSM1070905
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070905/suppl/GSM1070905_VFP-0021-6.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070906 | GPL201 |
|
VFP-0022_G4_DF [HG-Focus]
|
VFP_G4_DF
|
patient no.: 22
Stage: G4
infecting serotype: 1
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 4
scan date: 2007-11-09
|
|
| Sample_geo_accession | GSM1070906
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070906/suppl/GSM1070906_VFP-0022-1.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070907 | GPL201 |
|
VFP-0022_G6_DF [HG-Focus]
|
VFP_G6_DF
|
patient no.: 22
Stage: G6
infecting serotype: 1
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df1
scan date: 2007-11-09
|
|
| Sample_geo_accession | GSM1070907
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070907/suppl/GSM1070907_VFP-0022-5.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070908 | GPL201 |
|
VFP-0022_G7_DF [HG-Focus]
|
VFP_G7_DF
|
patient no.: 22
Stage: G7
infecting serotype: 1
phase: Convalenscent
severity: DF (dengue fever)
fever days or defervescent (df) days: c
scan date: 2007-11-09
|
|
| Sample_geo_accession | GSM1070908
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070908/suppl/GSM1070908_VFP-0022-6.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070909 | GPL201 |
|
VFP-0023_G3_DF [HG-Focus]
|
VFP_G3_DF
|
patient no.: 23
Stage: G3
infecting serotype: 1
phase: Early Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 3
scan date: 2008-01-18
|
|
| Sample_geo_accession | GSM1070909
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070909/suppl/GSM1070909_VFP-0023-1.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070910 | GPL201 |
|
VFP-0023_G6_DF [HG-Focus]
|
VFP_G6_DF
|
patient no.: 23
Stage: G6
infecting serotype: 1
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df1
scan date: 2008-01-18
|
|
| Sample_geo_accession | GSM1070910
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070910/suppl/GSM1070910_VFP-0023-5.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070911 | GPL201 |
|
VFP-0023_G7_DF [HG-Focus]
|
VFP_G7_DF
|
patient no.: 23
Stage: G7
infecting serotype: 1
phase: Convalenscent
severity: DF (dengue fever)
fever days or defervescent (df) days: c
scan date: 2008-01-18
|
|
| Sample_geo_accession | GSM1070911
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070911/suppl/GSM1070911_VFP-0023-6.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070912 | GPL201 |
|
VFP-0025_G4_DF [HG-Focus]
|
VFP_G4_DF
|
patient no.: 25
Stage: G4
infecting serotype: 3
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 4
scan date: 2007-11-09
|
|
| Sample_geo_accession | GSM1070912
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070912/suppl/GSM1070912_VFP-0025-1.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070913 | GPL201 |
|
VFP-0025_G6_DF [HG-Focus]
|
VFP_G6_DF
|
patient no.: 25
Stage: G6
infecting serotype: 3
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df0
scan date: 2007-11-09
|
|
| Sample_geo_accession | GSM1070913
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070913/suppl/GSM1070913_VFP-0025-5.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070914 | GPL201 |
|
VFP-0025_G7_DF [HG-Focus]
|
VFP_G7_DF
|
patient no.: 25
Stage: G7
infecting serotype: 3
phase: Convalenscent
severity: DF (dengue fever)
fever days or defervescent (df) days: c
scan date: 2007-11-09
|
|
| Sample_geo_accession | GSM1070914
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070914/suppl/GSM1070914_VFP-0025-6.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070915 | GPL201 |
|
VFP-0026_G2_DF [HG-Focus]
|
VFP_G2_DF
|
patient no.: 26
Stage: G2
infecting serotype: 1
phase: Early Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 2
scan date: 2008-01-18
|
|
| Sample_geo_accession | GSM1070915
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070915/suppl/GSM1070915_VFP-0026-1.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070916 | GPL201 |
|
VFP-0026_G6_DF [HG-Focus]
|
VFP_G6_DF
|
patient no.: 26
Stage: G6
infecting serotype: 1
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df1
scan date: 2008-01-18
|
|
| Sample_geo_accession | GSM1070916
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070916/suppl/GSM1070916_VFP-0026-5.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070917 | GPL201 |
|
VFP-0026_G7_DF [HG-Focus]
|
VFP_G7_DF
|
patient no.: 26
Stage: G7
infecting serotype: 1
phase: Convalenscent
severity: DF (dengue fever)
fever days or defervescent (df) days: c
scan date: 2008-01-18
|
|
| Sample_geo_accession | GSM1070917
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070917/suppl/GSM1070917_VFP-0026-6.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070918 | GPL201 |
|
VFP-0027_G2_DF [HG-Focus]
|
VFP_G2_DF
|
patient no.: 27
Stage: G2
infecting serotype: 1
phase: Early Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 2
scan date: 2008-01-18
|
|
| Sample_geo_accession | GSM1070918
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070918/suppl/GSM1070918_VFP-0027-1.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070919 | GPL201 |
|
VFP-0027_G6_DF [HG-Focus]
|
VFP_G6_DF
|
patient no.: 27
Stage: G6
infecting serotype: 1
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df1
scan date: 2008-01-18
|
|
| Sample_geo_accession | GSM1070919
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070919/suppl/GSM1070919_VFP-0027-5.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070920 | GPL201 |
|
VFP-0027_G7_DF [HG-Focus]
|
VFP_G7_DF
|
patient no.: 27
Stage: G7
infecting serotype: 1
phase: Convalenscent
severity: DF (dengue fever)
fever days or defervescent (df) days: c
scan date: 2008-01-18
|
|
| Sample_geo_accession | GSM1070920
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070920/suppl/GSM1070920_VFP-0027-6.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070921 | GPL201 |
|
VFP-0029_G6_DF [HG-Focus]
|
VFP_G6_DF
|
patient no.: 29
Stage: G6
infecting serotype: 1
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df2
scan date: 2008-01-18
|
|
| Sample_geo_accession | GSM1070921
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070921/suppl/GSM1070921_VFP-0029-5.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070922 | GPL201 |
|
VFP-0029_G7_DF [HG-Focus]
|
VFP_G7_DF
|
patient no.: 29
Stage: G7
infecting serotype: 1
phase: Convalenscent
severity: DF (dengue fever)
fever days or defervescent (df) days: c
scan date: 2008-01-18
|
|
| Sample_geo_accession | GSM1070922
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070922/suppl/GSM1070922_VFP-0029-6.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070923 | GPL201 |
|
VFP-0030_G2_DF [HG-Focus]
|
VFP_G2_DF
|
patient no.: 30
Stage: G2
infecting serotype: 1
phase: Early Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 2
scan date: 2007-11-09
|
|
| Sample_geo_accession | GSM1070923
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070923/suppl/GSM1070923_VFP-0030-1.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070924 | GPL201 |
|
VFP-0030_G5_DF [HG-Focus]
|
VFP_G5_DF
|
patient no.: 30
Stage: G5
infecting serotype: 1
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df1
scan date: 2007-11-09
|
|
| Sample_geo_accession | GSM1070924
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070924/suppl/GSM1070924_VFP-0030-5.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070925 | GPL201 |
|
VFP-0030_G7_DF [HG-Focus]
|
VFP_G7_DF
|
patient no.: 30
Stage: G7
infecting serotype: 1
phase: Convalenscent
severity: DF (dengue fever)
fever days or defervescent (df) days: c
scan date: 2007-11-09
|
|
| Sample_geo_accession | GSM1070925
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070925/suppl/GSM1070925_VFP-0030-6.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070926 | GPL201 |
|
VFP-0031_G4_DF [HG-Focus]
|
VFP_G4_DF
|
patient no.: 31
Stage: G4
infecting serotype: 4
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df0
scan date: 2008-01-16
|
|
| Sample_geo_accession | GSM1070926
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070926/suppl/GSM1070926_VFP-0031-1.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070927 | GPL201 |
|
VFP-0031_G5_DF [HG-Focus]
|
VFP_G5_DF
|
patient no.: 31
Stage: G5
infecting serotype: 4
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df1
scan date: 2008-01-16
|
|
| Sample_geo_accession | GSM1070927
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070927/suppl/GSM1070927_VFP-0031-5.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070928 | GPL201 |
|
VFP-0031_G7_DF [HG-Focus]
|
VFP_G7_DF
|
patient no.: 31
Stage: G7
infecting serotype: 4
phase: Convalenscent
severity: DF (dengue fever)
fever days or defervescent (df) days: c
scan date: 2008-01-16
|
|
| Sample_geo_accession | GSM1070928
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070928/suppl/GSM1070928_VFP-0031-6.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070929 | GPL201 |
|
VFP-0033_G2_DF [HG-Focus]
|
VFP_G2_DF
|
patient no.: 33
Stage: G2
infecting serotype: 4
phase: Early Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 2
scan date: 2007-11-15
|
|
| Sample_geo_accession | GSM1070929
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070929/suppl/GSM1070929_VFP-0033-1.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070930 | GPL201 |
|
VFP-0033_G5_DF [HG-Focus]
|
VFP_G5_DF
|
patient no.: 33
Stage: G5
infecting serotype: 4
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df1
scan date: 2007-11-15
|
|
| Sample_geo_accession | GSM1070930
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070930/suppl/GSM1070930_VFP-0033-5.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070931 | GPL201 |
|
VFP-0033_G7_DF [HG-Focus]
|
VFP_G7_DF
|
patient no.: 33
Stage: G7
infecting serotype: 4
phase: Convalenscent
severity: DF (dengue fever)
fever days or defervescent (df) days: c
scan date: 2007-11-15
|
|
| Sample_geo_accession | GSM1070931
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070931/suppl/GSM1070931_VFP-0033-6.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070932 | GPL201 |
|
VFP-0034_G3_DF [HG-Focus]
|
VFP_G3_DF
|
patient no.: 34
Stage: G3
infecting serotype: 1
phase: Early Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 2
scan date: 2007-11-15
|
|
| Sample_geo_accession | GSM1070932
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070932/suppl/GSM1070932_VFP-0034-1.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070933 | GPL201 |
|
VFP-0034_G6_DF [HG-Focus]
|
VFP_G6_DF
|
patient no.: 34
Stage: G6
infecting serotype: 1
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df1
scan date: 2007-11-15
|
|
| Sample_geo_accession | GSM1070933
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070933/suppl/GSM1070933_VFP-0034-5.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070934 | GPL201 |
|
VFP-0034_G7_DF [HG-Focus]
|
VFP_G7_DF
|
patient no.: 34
Stage: G7
infecting serotype: 1
phase: Convalenscent
severity: DF (dengue fever)
fever days or defervescent (df) days: c
scan date: 2007-11-15
|
|
| Sample_geo_accession | GSM1070934
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070934/suppl/GSM1070934_VFP-0034-6.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070935 | GPL201 |
|
VFP-0037_G4_DF [HG-Focus]
|
VFP_G4_DF
|
patient no.: 37
Stage: G4
infecting serotype: 3
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 4
scan date: 2007-11-28
|
|
| Sample_geo_accession | GSM1070935
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070935/suppl/GSM1070935_VFP-0037-1.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070936 | GPL201 |
|
VFP-0037_G6_DF [HG-Focus]
|
VFP_G6_DF
|
patient no.: 37
Stage: G6
infecting serotype: 3
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df1
scan date: 2007-11-28
|
|
| Sample_geo_accession | GSM1070936
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070936/suppl/GSM1070936_VFP-0037-5.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070937 | GPL201 |
|
VFP-0037_G7_DF [HG-Focus]
|
VFP_G7_DF
|
patient no.: 37
Stage: G7
infecting serotype: 3
phase: Convalenscent
severity: DF (dengue fever)
fever days or defervescent (df) days: c
scan date: 2007-11-28
|
|
| Sample_geo_accession | GSM1070937
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070937/suppl/GSM1070937_VFP-0037-6.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070938 | GPL201 |
|
VFP-0039_G2_DF [HG-Focus]
|
VFP_G2_DF
|
patient no.: 39
Stage: G2
infecting serotype: 1
phase: Early Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 2
scan date: 2007-11-28
|
|
| Sample_geo_accession | GSM1070938
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070938/suppl/GSM1070938_VFP-0039-1.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070939 | GPL201 |
|
VFP-0039_G5_DF [HG-Focus]
|
VFP_G5_DF
|
patient no.: 39
Stage: G5
infecting serotype: 1
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df1
scan date: 2007-11-28
|
|
| Sample_geo_accession | GSM1070939
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070939/suppl/GSM1070939_VFP-0039-5.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070940 | GPL201 |
|
VFP-0039_G7_DF [HG-Focus]
|
VFP_G7_DF
|
patient no.: 39
Stage: G7
infecting serotype: 1
phase: Convalenscent
severity: DF (dengue fever)
fever days or defervescent (df) days: c
scan date: 2007-11-28
|
|
| Sample_geo_accession | GSM1070940
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070940/suppl/GSM1070940_VFP-0039-6.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070941 | GPL201 |
|
VFP-0040_G2_DF [HG-Focus]
|
VFP_G2_DF
|
patient no.: 40
Stage: G2
infecting serotype: 3
phase: Early Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 2
scan date: 2007-11-30
|
|
| Sample_geo_accession | GSM1070941
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070941/suppl/GSM1070941_VFP-0040-1.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070942 | GPL201 |
|
VFP-0040_G4_DF [HG-Focus]
|
VFP_G4_DF
|
patient no.: 40
Stage: G4
infecting serotype: 3
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df0
scan date: 2007-11-30
|
|
| Sample_geo_accession | GSM1070942
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070942/suppl/GSM1070942_VFP-0040-5.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070943 | GPL201 |
|
VFP-0040_G6_DF [HG-Focus]
|
VFP_G6_DF
|
patient no.: 40
Stage: G6
infecting serotype: 3
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df2
scan date: 2007-11-30
|
|
| Sample_geo_accession | GSM1070943
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070943/suppl/GSM1070943_VFP-0040-6.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070944 | GPL201 |
|
VFP-0041_G3_DF [HG-Focus]
|
VFP_G3_DF
|
patient no.: 41
Stage: G3
infecting serotype: 1
phase: Early Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 3
scan date: 2008-01-09
|
|
| Sample_geo_accession | GSM1070944
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070944/suppl/GSM1070944_VFP-0041-1.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070945 | GPL201 |
|
VFP-0041_G4_DF [HG-Focus]
|
VFP_G4_DF
|
patient no.: 41
Stage: G4
infecting serotype: 1
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 4
scan date: 2008-01-09
|
|
| Sample_geo_accession | GSM1070945
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070945/suppl/GSM1070945_VFP-0041-2.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070946 | GPL201 |
|
VFP-0041_G5_DF [HG-Focus]
|
VFP_G5_DF
|
patient no.: 41
Stage: G5
infecting serotype: 1
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 5
scan date: 2008-01-09
|
|
| Sample_geo_accession | GSM1070946
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070946/suppl/GSM1070946_VFP-0041-3.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070947 | GPL201 |
|
VFP-0041_G6(1)_DF [HG-Focus]
|
VFP_G6_DF
|
patient no.: 41
Stage: G6
infecting serotype: 1
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df0
scan date: 2008-01-09
|
|
| Sample_geo_accession | GSM1070947
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070947/suppl/GSM1070947_VFP-0041-4.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070948 | GPL201 |
|
VFP-0041_G6(2)_DF [HG-Focus]
|
VFP_G6_DF
|
patient no.: 41
Stage: G6
infecting serotype: 1
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 6
scan date: 2008-01-09
|
|
| Sample_geo_accession | GSM1070948
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070948/suppl/GSM1070948_VFP-0041-5.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070949 | GPL201 |
|
VFP-0041_G7_DF [HG-Focus]
|
VFP_G7_DF
|
patient no.: 41
Stage: G7
infecting serotype: 1
phase: Convalenscent
severity: DF (dengue fever)
fever days or defervescent (df) days: c
scan date: 2008-01-09
|
|
| Sample_geo_accession | GSM1070949
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070949/suppl/GSM1070949_VFP-0041-6.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070950 | GPL201 |
|
VFP-0045_G4_DF [HG-Focus]
|
VFP_G4_DF
|
patient no.: 45
Stage: G4
infecting serotype: 1
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 4
scan date: 2007-11-30
|
|
| Sample_geo_accession | GSM1070950
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070950/suppl/GSM1070950_VFP-0045-1.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070951 | GPL201 |
|
VFP-0045_G5_DF [HG-Focus]
|
VFP_G5_DF
|
patient no.: 45
Stage: G5
infecting serotype: 1
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df0
scan date: 2007-11-30
|
|
| Sample_geo_accession | GSM1070951
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070951/suppl/GSM1070951_VFP-0045-5.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070952 | GPL201 |
|
VFP-0045_G7_DF [HG-Focus]
|
VFP_G7_DF
|
patient no.: 45
Stage: G7
infecting serotype: 1
phase: Convalenscent
severity: DF (dengue fever)
fever days or defervescent (df) days: c
scan date: 2007-11-30
|
|
| Sample_geo_accession | GSM1070952
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070952/suppl/GSM1070952_VFP-0045-6.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070953 | GPL201 |
|
VFP-0047_G4_DHF [HG-Focus]
|
VFP_G4_DHF
|
patient no.: 47
Stage: G4
infecting serotype: 1
phase: Late Acute
severity: DHF (dengue hemorrhagic fever)
fever days or defervescent (df) days: 4
scan date: 2007-11-30
|
|
| Sample_geo_accession | GSM1070953
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070953/suppl/GSM1070953_VFP-0047-1.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070954 | GPL201 |
|
VFP-0047_G5_DHF [HG-Focus]
|
VFP_G5_DHF
|
patient no.: 47
Stage: G5
infecting serotype: 1
phase: Late Acute
severity: DHF (dengue hemorrhagic fever)
fever days or defervescent (df) days: df0
scan date: 2007-11-30
|
|
| Sample_geo_accession | GSM1070954
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070954/suppl/GSM1070954_VFP-0047-5.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070955 | GPL201 |
|
VFP-0047_G7_DHF [HG-Focus]
|
VFP_G7_DHF
|
patient no.: 47
Stage: G7
infecting serotype: 1
phase: Convalenscent
severity: DHF (dengue hemorrhagic fever)
fever days or defervescent (df) days: c
scan date: 2007-11-30
|
|
| Sample_geo_accession | GSM1070955
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070955/suppl/GSM1070955_VFP-0047-6.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070956 | GPL201 |
|
VFP-0053_G3_DF [HG-Focus]
|
VFP_G3_DF
|
patient no.: 53
Stage: G3
infecting serotype: 2
phase: Early Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 3
scan date: 2007-11-30
|
|
| Sample_geo_accession | GSM1070956
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070956/suppl/GSM1070956_VFP-0053-1.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070957 | GPL201 |
|
VFP-0053_G4_DF [HG-Focus]
|
VFP_G4_DF
|
patient no.: 53
Stage: G4
infecting serotype: 2
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df0
scan date: 2007-11-30
|
|
| Sample_geo_accession | GSM1070957
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070957/suppl/GSM1070957_VFP-0053-5.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070958 | GPL201 |
|
VFP-0053_G7_DF [HG-Focus]
|
VFP_G7_DF
|
patient no.: 53
Stage: G7
infecting serotype: 2
phase: Convalenscent
severity: DF (dengue fever)
fever days or defervescent (df) days: c
scan date: 2007-11-30
|
|
| Sample_geo_accession | GSM1070958
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070958/suppl/GSM1070958_VFP-0053-6.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070959 | GPL201 |
|
VFP-0056_G2_DF [HG-Focus]
|
VFP_G2_DF
|
patient no.: 56
Stage: G2
infecting serotype: 1
phase: Early Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 2
scan date: 2008-01-09
|
|
| Sample_geo_accession | GSM1070959
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070959/suppl/GSM1070959_VFP-0056-1.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070960 | GPL201 |
|
VFP-0056_G3_DF [HG-Focus]
|
VFP_G3_DF
|
patient no.: 56
Stage: G3
infecting serotype: 1
phase: Early Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 3
scan date: 2008-01-09
|
|
| Sample_geo_accession | GSM1070960
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070960/suppl/GSM1070960_VFP-0056-2.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070961 | GPL201 |
|
VFP-0056_G4_DF [HG-Focus]
|
VFP_G4_DF
|
patient no.: 56
Stage: G4
infecting serotype: 1
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df0
scan date: 2008-01-09
|
|
| Sample_geo_accession | GSM1070961
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070961/suppl/GSM1070961_VFP-0056-3.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070962 | GPL201 |
|
VFP-0056_G5_DF [HG-Focus]
|
VFP_G5_DF
|
patient no.: 56
Stage: G5
infecting serotype: 1
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df1
scan date: 2008-01-09
|
|
| Sample_geo_accession | GSM1070962
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070962/suppl/GSM1070962_VFP-0056-4.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070963 | GPL201 |
|
VFP-0056_G6_DF [HG-Focus]
|
VFP_G6_DF
|
patient no.: 56
Stage: G6
infecting serotype: 1
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df4
scan date: 2008-01-09
|
|
| Sample_geo_accession | GSM1070963
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070963/suppl/GSM1070963_VFP-0056-5.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070964 | GPL201 |
|
VFP-0056_G7_DF [HG-Focus]
|
VFP_G7_DF
|
patient no.: 56
Stage: G7
infecting serotype: 1
phase: Convalenscent
severity: DF (dengue fever)
fever days or defervescent (df) days: c
scan date: 2008-01-09
|
|
| Sample_geo_accession | GSM1070964
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070964/suppl/GSM1070964_VFP-0056-6.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070965 | GPL201 |
|
VFP-0057_G1_DF [HG-Focus]
|
VFP_G1_DF
|
patient no.: 57
Stage: G1
infecting serotype: 1
phase: Early Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 1
scan date: 2008-01-11
|
|
| Sample_geo_accession | GSM1070965
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070965/suppl/GSM1070965_VFP-0057-1.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070966 | GPL201 |
|
VFP-0057_G2_DF [HG-Focus]
|
VFP_G2_DF
|
patient no.: 57
Stage: G2
infecting serotype: 1
phase: Early Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 2
scan date: 2008-01-11
|
|
| Sample_geo_accession | GSM1070966
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070966/suppl/GSM1070966_VFP-0057-2.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070967 | GPL201 |
|
VFP-0057_G3_DF [HG-Focus]
|
VFP_G3_DF
|
patient no.: 57
Stage: G3
infecting serotype: 1
phase: Early Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 3
scan date: 2008-01-11
|
|
| Sample_geo_accession | GSM1070967
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070967/suppl/GSM1070967_VFP-0057-3.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070968 | GPL201 |
|
VFP-0057_G4_DF [HG-Focus]
|
VFP_G4_DF
|
patient no.: 57
Stage: G4
infecting serotype: 1
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df0
scan date: 2008-01-11
|
|
| Sample_geo_accession | GSM1070968
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070968/suppl/GSM1070968_VFP-0057-4.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070969 | GPL201 |
|
VFP-0057_G6_DF [HG-Focus]
|
VFP_G6_DF
|
patient no.: 57
Stage: G6
infecting serotype: 1
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df3
scan date: 2008-01-11
|
|
| Sample_geo_accession | GSM1070969
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070969/suppl/GSM1070969_VFP-0057-5.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070970 | GPL201 |
|
VFP-0057_G7_DF [HG-Focus]
|
VFP_G7_DF
|
patient no.: 57
Stage: G7
infecting serotype: 1
phase: Convalenscent
severity: DF (dengue fever)
fever days or defervescent (df) days: c
scan date: 2008-01-11
|
|
| Sample_geo_accession | GSM1070970
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070970/suppl/GSM1070970_VFP-0057-6.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070971 | GPL201 |
|
VFP-0060_G3_DHF [HG-Focus]
|
VFP_G3_DHF
|
patient no.: 60
Stage: G3
infecting serotype: 2
phase: Early Acute
severity: DHF (dengue hemorrhagic fever)
fever days or defervescent (df) days: 3
scan date: 2007-12-05
|
|
| Sample_geo_accession | GSM1070971
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070971/suppl/GSM1070971_VFP-0060-1.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070972 | GPL201 |
|
VFP-0060_G4_DHF [HG-Focus]
|
VFP_G4_DHF
|
patient no.: 60
Stage: G4
infecting serotype: 2
phase: Late Acute
severity: DHF (dengue hemorrhagic fever)
fever days or defervescent (df) days: 4
scan date: 2007-12-05
|
|
| Sample_geo_accession | GSM1070972
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070972/suppl/GSM1070972_VFP-0060-5.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070973 | GPL201 |
|
VFP-0060_G7_DHF [HG-Focus]
|
VFP_G7_DHF
|
patient no.: 60
Stage: G7
infecting serotype: 2
phase: Convalenscent
severity: DHF (dengue hemorrhagic fever)
fever days or defervescent (df) days: c
scan date: 2007-12-05
|
|
| Sample_geo_accession | GSM1070973
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070973/suppl/GSM1070973_VFP-0060-6.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070974 | GPL201 |
|
VFP-0063_G3_DF [HG-Focus]
|
VFP_G3_DF
|
patient no.: 63
Stage: G3
infecting serotype: 3
phase: Early Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 3
scan date: 2007-12-05
|
|
| Sample_geo_accession | GSM1070974
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070974/suppl/GSM1070974_VFP-0063-1.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070975 | GPL201 |
|
VFP-0063_G5_DF [HG-Focus]
|
VFP_G5_DF
|
patient no.: 63
Stage: G5
infecting serotype: 3
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df1
scan date: 2007-12-05
|
|
| Sample_geo_accession | GSM1070975
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070975/suppl/GSM1070975_VFP-0063-5.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070976 | GPL201 |
|
VFP-0063_G7_DF [HG-Focus]
|
VFP_G7_DF
|
patient no.: 63
Stage: G7
infecting serotype: 3
phase: Convalenscent
severity: DF (dengue fever)
fever days or defervescent (df) days: c
scan date: 2007-12-05
|
|
| Sample_geo_accession | GSM1070976
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070976/suppl/GSM1070976_VFP-0063-6.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070977 | GPL201 |
|
VFP-0064_G2_DF [HG-Focus]
|
VFP_G2_DF
|
patient no.: 64
Stage: G2
infecting serotype: 1
phase: Early Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 2
scan date: 2008-01-11
|
|
| Sample_geo_accession | GSM1070977
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070977/suppl/GSM1070977_VFP-0064-1.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070978 | GPL201 |
|
VFP-0064_G3_DF [HG-Focus]
|
VFP_G3_DF
|
patient no.: 64
Stage: G3
infecting serotype: 1
phase: Early Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 3
scan date: 2008-01-11
|
|
| Sample_geo_accession | GSM1070978
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070978/suppl/GSM1070978_VFP-0064-2.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070979 | GPL201 |
|
VFP-0064_G4_DF [HG-Focus]
|
VFP_G4_DF
|
patient no.: 64
Stage: G4
infecting serotype: 1
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 4
scan date: 2008-01-11
|
|
| Sample_geo_accession | GSM1070979
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070979/suppl/GSM1070979_VFP-0064-3.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070980 | GPL201 |
|
VFP-0064_G5_DF [HG-Focus]
|
VFP_G5_DF
|
patient no.: 64
Stage: G5
infecting serotype: 1
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 5
scan date: 2008-01-11
|
|
| Sample_geo_accession | GSM1070980
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070980/suppl/GSM1070980_VFP-0064-4.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070981 | GPL201 |
|
VFP-0064_G6_DF [HG-Focus]
|
VFP_G6_DF
|
patient no.: 64
Stage: G6
infecting serotype: 1
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df0
scan date: 2008-01-11
|
|
| Sample_geo_accession | GSM1070981
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070981/suppl/GSM1070981_VFP-0064-5.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070982 | GPL201 |
|
VFP-0064_G7_DF [HG-Focus]
|
VFP_G7_DF
|
patient no.: 64
Stage: G7
infecting serotype: 1
phase: Convalenscent
severity: DF (dengue fever)
fever days or defervescent (df) days: c
scan date: 2008-01-11
|
|
| Sample_geo_accession | GSM1070982
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070982/suppl/GSM1070982_VFP-0064-6.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070983 | GPL201 |
|
VFP-0070_G2_DF [HG-Focus]
|
VFP_G2_DF
|
patient no.: 70
Stage: G2
infecting serotype: 2
phase: Early Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 2
scan date: 2007-12-05
|
|
| Sample_geo_accession | GSM1070983
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070983/suppl/GSM1070983_VFP-0070-1.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070984 | GPL201 |
|
VFP-0070_G4_DF [HG-Focus]
|
VFP_G4_DF
|
patient no.: 70
Stage: G4
infecting serotype: 2
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df1
scan date: 2007-12-05
|
|
| Sample_geo_accession | GSM1070984
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070984/suppl/GSM1070984_VFP-0070-5.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070985 | GPL201 |
|
VFP-0070_G7_DF [HG-Focus]
|
VFP_G7_DF
|
patient no.: 70
Stage: G7
infecting serotype: 2
phase: Convalenscent
severity: DF (dengue fever)
fever days or defervescent (df) days: c
scan date: 2007-12-05
|
|
| Sample_geo_accession | GSM1070985
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070985/suppl/GSM1070985_VFP-0070-6.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070986 | GPL201 |
|
VFP-0075_G2_DF [HG-Focus]
|
VFP_G2_DF
|
patient no.: 75
Stage: G2
infecting serotype: 2
phase: Early Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 2
scan date: 2007-12-05
|
|
| Sample_geo_accession | GSM1070986
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070986/suppl/GSM1070986_VFP-0075-1.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070987 | GPL201 |
|
VFP-0075_G5_DF [HG-Focus]
|
VFP_G5_DF
|
patient no.: 75
Stage: G5
infecting serotype: 2
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df2
scan date: 2007-12-05
|
|
| Sample_geo_accession | GSM1070987
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070987/suppl/GSM1070987_VFP-0075-5.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070988 | GPL201 |
|
VFP-0075_G7_DF [HG-Focus]
|
VFP_G7_DF
|
patient no.: 75
Stage: G7
infecting serotype: 2
phase: Convalenscent
severity: DF (dengue fever)
fever days or defervescent (df) days: c
scan date: 2007-12-05
|
|
| Sample_geo_accession | GSM1070988
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070988/suppl/GSM1070988_VFP-0075-6.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070989 | GPL201 |
|
VFP-0081_G2_DF [HG-Focus]
|
VFP_G2_DF
|
patient no.: 81
Stage: G2
infecting serotype: 1
phase: Early Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 2
scan date: 2007-12-07
|
|
| Sample_geo_accession | GSM1070989
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070989/suppl/GSM1070989_VFP-0081-1.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070990 | GPL201 |
|
VFP-0081_G6_DF [HG-Focus]
|
VFP_G6_DF
|
patient no.: 81
Stage: G6
infecting serotype: 1
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df1
scan date: 2007-12-07
|
|
| Sample_geo_accession | GSM1070990
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070990/suppl/GSM1070990_VFP-0081-5.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070991 | GPL201 |
|
VFP-0081_G7_DF [HG-Focus]
|
VFP_G7_DF
|
patient no.: 81
Stage: G7
infecting serotype: 1
phase: Convalenscent
severity: DF (dengue fever)
fever days or defervescent (df) days: c
scan date: 2007-12-07
|
|
| Sample_geo_accession | GSM1070991
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070991/suppl/GSM1070991_VFP-0081-6.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070992 | GPL201 |
|
VFP-0082_G4_DF [HG-Focus]
|
VFP_G4_DF
|
patient no.: 82
Stage: G4
infecting serotype: 1
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 4
scan date: 2007-12-07
|
|
| Sample_geo_accession | GSM1070992
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070992/suppl/GSM1070992_VFP-0082-1.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070993 | GPL201 |
|
VFP-0082_G5_DF [HG-Focus]
|
VFP_G5_DF
|
patient no.: 82
Stage: G5
infecting serotype: 1
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df0
scan date: 2007-12-07
|
|
| Sample_geo_accession | GSM1070993
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070993/suppl/GSM1070993_VFP-0082-5.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070994 | GPL201 |
|
VFP-0082_G7_DF [HG-Focus]
|
VFP_G7_DF
|
patient no.: 82
Stage: G7
infecting serotype: 1
phase: Convalenscent
severity: DF (dengue fever)
fever days or defervescent (df) days: c
scan date: 2007-12-07
|
|
| Sample_geo_accession | GSM1070994
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070994/suppl/GSM1070994_VFP-0082-6.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070995 | GPL201 |
|
VFP-0083_G2_DF [HG-Focus]
|
VFP_G2_DF
|
patient no.: 83
Stage: G2
infecting serotype: 1
phase: Early Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 2
scan date: 2007-12-07
|
|
| Sample_geo_accession | GSM1070995
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070995/suppl/GSM1070995_VFP-0083-1.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070996 | GPL201 |
|
VFP-0083_G5_DF [HG-Focus]
|
VFP_G5_DF
|
patient no.: 83
Stage: G5
infecting serotype: 1
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df2
scan date: 2007-12-07
|
|
| Sample_geo_accession | GSM1070996
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070996/suppl/GSM1070996_VFP-0083-5.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070997 | GPL201 |
|
VFP-0083_G7_DF [HG-Focus]
|
VFP_G7_DF
|
patient no.: 83
Stage: G7
infecting serotype: 1
phase: Convalenscent
severity: DF (dengue fever)
fever days or defervescent (df) days: c
scan date: 2007-12-07
|
|
| Sample_geo_accession | GSM1070997
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070997/suppl/GSM1070997_VFP-0083-6.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070998 | GPL201 |
|
VFP-0085_G3_DF [HG-Focus]
|
VFP_G3_DF
|
patient no.: 85
Stage: G3
infecting serotype: 3
phase: Early Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 3
scan date: 2007-12-07
|
|
| Sample_geo_accession | GSM1070998
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070998/suppl/GSM1070998_VFP-0085-1.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1070999 | GPL201 |
|
VFP-0085_G5_DF [HG-Focus]
|
VFP_G5_DF
|
patient no.: 85
Stage: G5
infecting serotype: 3
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df1
scan date: 2007-12-07
|
|
| Sample_geo_accession | GSM1070999
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1070nnn/GSM1070999/suppl/GSM1070999_VFP-0085-5.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1071000 | GPL201 |
|
VFP-0085_G7_DF [HG-Focus]
|
VFP_G7_DF
|
patient no.: 85
Stage: G7
infecting serotype: 3
phase: Convalenscent
severity: DF (dengue fever)
fever days or defervescent (df) days: c
scan date: 2007-12-07
|
|
| Sample_geo_accession | GSM1071000
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1071nnn/GSM1071000/suppl/GSM1071000_VFP-0085-6.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1071001 | GPL201 |
|
VFP-0087_G3_DF [HG-Focus]
|
VFP_G3_DF
|
patient no.: 87
Stage: G3
phase: Early Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 3
scan date: 2007-12-12
|
|
| Sample_geo_accession | GSM1071001
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1071nnn/GSM1071001/suppl/GSM1071001_VFP-0087-1.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1071002 | GPL201 |
|
VFP-0087_G5_DF [HG-Focus]
|
VFP_G5_DF
|
patient no.: 87
Stage: G5
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df1
scan date: 2007-12-12
|
|
| Sample_geo_accession | GSM1071002
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1071nnn/GSM1071002/suppl/GSM1071002_VFP-0087-5.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1071003 | GPL201 |
|
VFP-0087_G7_DF [HG-Focus]
|
VFP_G7_DF
|
patient no.: 87
Stage: G7
phase: Convalenscent
severity: DF (dengue fever)
fever days or defervescent (df) days: c
scan date: 2007-12-12
|
|
| Sample_geo_accession | GSM1071003
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1071nnn/GSM1071003/suppl/GSM1071003_VFP-0087-6.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1071004 | GPL201 |
|
VFP-0090_G2_DF [HG-Focus]
|
VFP_G2_DF
|
patient no.: 90
Stage: G2
infecting serotype: 4
phase: Early Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 2
scan date: 2007-12-12
|
|
| Sample_geo_accession | GSM1071004
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1071nnn/GSM1071004/suppl/GSM1071004_VFP-0090-1.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1071005 | GPL201 |
|
VFP-0090_G5_DF [HG-Focus]
|
VFP_G5_DF
|
patient no.: 90
Stage: G5
infecting serotype: 4
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df1
scan date: 2007-12-12
|
|
| Sample_geo_accession | GSM1071005
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1071nnn/GSM1071005/suppl/GSM1071005_VFP-0090-5.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1071006 | GPL201 |
|
VFP-0090_G7_DF [HG-Focus]
|
VFP_G7_DF
|
patient no.: 90
Stage: G7
infecting serotype: 4
phase: Convalenscent
severity: DF (dengue fever)
fever days or defervescent (df) days: c
scan date: 2007-12-12
|
|
| Sample_geo_accession | GSM1071006
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1071nnn/GSM1071006/suppl/GSM1071006_VFP-0090-6.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1071007 | GPL201 |
|
VFP-0101_G3_DF [HG-Focus]
|
VFP_G3_DF
|
patient no.: 101
Stage: G3
infecting serotype: 2
phase: Early Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 3
scan date: 2007-12-12
|
|
| Sample_geo_accession | GSM1071007
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1071nnn/GSM1071007/suppl/GSM1071007_VFP-0101-1.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1071008 | GPL201 |
|
VFP-0101_G4_DF [HG-Focus]
|
VFP_G4_DF
|
patient no.: 101
Stage: G4
infecting serotype: 2
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df1
scan date: 2007-12-12
|
|
| Sample_geo_accession | GSM1071008
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1071nnn/GSM1071008/suppl/GSM1071008_VFP-0101-5.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1071009 | GPL201 |
|
VFP-0101_G7_DF [HG-Focus]
|
VFP_G7_DF
|
patient no.: 101
Stage: G7
infecting serotype: 2
phase: Convalenscent
severity: DF (dengue fever)
fever days or defervescent (df) days: c
scan date: 2007-12-12
|
|
| Sample_geo_accession | GSM1071009
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1071nnn/GSM1071009/suppl/GSM1071009_VFP-0101-6.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1071010 | GPL201 |
|
VFP-0104_G3_DF [HG-Focus]
|
VFP_G3_DF
|
patient no.: 104
Stage: G3
infecting serotype: 3
phase: Early Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 3
scan date: 2007-12-12
|
|
| Sample_geo_accession | GSM1071010
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1071nnn/GSM1071010/suppl/GSM1071010_VFP-0104-1.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1071011 | GPL201 |
|
VFP-0104_G6_DF [HG-Focus]
|
VFP_G6_DF
|
patient no.: 104
Stage: G6
infecting serotype: 3
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df2
scan date: 2007-12-12
|
|
| Sample_geo_accession | GSM1071011
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1071nnn/GSM1071011/suppl/GSM1071011_VFP-0104-5.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1071012 | GPL201 |
|
VFP-0104_G7_DF [HG-Focus]
|
VFP_G7_DF
|
patient no.: 104
Stage: G7
infecting serotype: 3
phase: Convalenscent
severity: DF (dengue fever)
fever days or defervescent (df) days: c
scan date: 2007-12-12
|
|
| Sample_geo_accession | GSM1071012
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1071nnn/GSM1071012/suppl/GSM1071012_VFP-0104-6.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1071013 | GPL201 |
|
VFP-0105_G3_DF [HG-Focus]
|
VFP_G3_DF
|
patient no.: 105
Stage: G3
infecting serotype: 4
phase: Early Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 3
scan date: 2008-01-03
|
|
| Sample_geo_accession | GSM1071013
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1071nnn/GSM1071013/suppl/GSM1071013_VFP-0105-1.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1071014 | GPL201 |
|
VFP-0105_G4_DF [HG-Focus]
|
VFP_G4_DF
|
patient no.: 105
Stage: G4
infecting serotype: 4
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df3
scan date: 2008-01-03
|
|
| Sample_geo_accession | GSM1071014
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1071nnn/GSM1071014/suppl/GSM1071014_VFP-0105-5.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1071015 | GPL201 |
|
VFP-0105_G7_DF [HG-Focus]
|
VFP_G7_DF
|
patient no.: 105
Stage: G7
infecting serotype: 4
phase: Convalenscent
severity: DF (dengue fever)
fever days or defervescent (df) days: c
scan date: 2008-01-03
|
|
| Sample_geo_accession | GSM1071015
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1071nnn/GSM1071015/suppl/GSM1071015_VFP-0105-6.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1071016 | GPL201 |
|
VFP-0111_G4_DF [HG-Focus]
|
VFP_G4_DF
|
patient no.: 111
Stage: G4
infecting serotype: 4
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 4
scan date: 2008-01-25
|
|
| Sample_geo_accession | GSM1071016
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1071nnn/GSM1071016/suppl/GSM1071016_VFP-0111-2.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1071017 | GPL201 |
|
VFP-0111_G6_DF [HG-Focus]
|
VFP_G6_DF
|
patient no.: 111
Stage: G6
infecting serotype: 4
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df1
scan date: 2008-01-25
|
|
| Sample_geo_accession | GSM1071017
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1071nnn/GSM1071017/suppl/GSM1071017_VFP-0111-5.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1071018 | GPL201 |
|
VFP-0111_G7_DF [HG-Focus]
|
VFP_G7_DF
|
patient no.: 111
Stage: G7
infecting serotype: 4
phase: Convalenscent
severity: DF (dengue fever)
fever days or defervescent (df) days: c
scan date: 2008-01-25
|
|
| Sample_geo_accession | GSM1071018
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1071nnn/GSM1071018/suppl/GSM1071018_VFP-0111-6.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1071019 | GPL201 |
|
VFP-0113_G2_DF [HG-Focus]
|
VFP_G2_DF
|
patient no.: 113
Stage: G2
infecting serotype: 3
phase: Early Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 2
scan date: 2008-01-03
|
|
| Sample_geo_accession | GSM1071019
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1071nnn/GSM1071019/suppl/GSM1071019_VFP-0113-1.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1071020 | GPL201 |
|
VFP-0113_G6_DF [HG-Focus]
|
VFP_G6_DF
|
patient no.: 113
Stage: G6
infecting serotype: 3
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df1
scan date: 2008-01-03
|
|
| Sample_geo_accession | GSM1071020
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1071nnn/GSM1071020/suppl/GSM1071020_VFP-0113-5.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1071021 | GPL201 |
|
VFP-0113_G7_DF [HG-Focus]
|
VFP_G7_DF
|
patient no.: 113
Stage: G7
infecting serotype: 3
phase: Convalenscent
severity: DF (dengue fever)
fever days or defervescent (df) days: c
scan date: 2008-01-03
|
|
| Sample_geo_accession | GSM1071021
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1071nnn/GSM1071021/suppl/GSM1071021_VFP-0113-6.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1071022 | GPL201 |
|
VFP-0117_G4_DF [HG-Focus]
|
VFP_G4_DF
|
patient no.: 117
Stage: G4
infecting serotype: 3
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 4
scan date: 2008-01-04
|
|
| Sample_geo_accession | GSM1071022
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1071nnn/GSM1071022/suppl/GSM1071022_VFP-0117-1.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1071023 | GPL201 |
|
VFP-0117_G6_DF [HG-Focus]
|
VFP_G6_DF
|
patient no.: 117
Stage: G6
infecting serotype: 3
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df1
scan date: 2008-01-04
|
|
| Sample_geo_accession | GSM1071023
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1071nnn/GSM1071023/suppl/GSM1071023_VFP-0117-5.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1071024 | GPL201 |
|
VFP-0117_G7_DF [HG-Focus]
|
VFP_G7_DF
|
patient no.: 117
Stage: G7
infecting serotype: 3
phase: Convalenscent
severity: DF (dengue fever)
fever days or defervescent (df) days: c
scan date: 2008-01-04
|
|
| Sample_geo_accession | GSM1071024
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1071nnn/GSM1071024/suppl/GSM1071024_VFP-0117-6.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1071025 | GPL201 |
|
VFP-0129_G1_DF [HG-Focus]
|
VFP_G1_DF
|
patient no.: 129
Stage: G1
infecting serotype: 4
phase: Early Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 1
scan date: 2008-01-04
|
|
| Sample_geo_accession | GSM1071025
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1071nnn/GSM1071025/suppl/GSM1071025_VFP-0129-1.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1071026 | GPL201 |
|
VFP-0129_G4_DF [HG-Focus]
|
VFP_G4_DF
|
patient no.: 129
Stage: G4
infecting serotype: 4
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df2
scan date: 2008-01-04
|
|
| Sample_geo_accession | GSM1071026
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1071nnn/GSM1071026/suppl/GSM1071026_VFP-0129-5.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1071027 | GPL201 |
|
VFP-0129_G7_DF [HG-Focus]
|
VFP_G7_DF
|
patient no.: 129
Stage: G7
infecting serotype: 4
phase: Convalenscent
severity: DF (dengue fever)
fever days or defervescent (df) days: c
scan date: 2008-01-04
|
|
| Sample_geo_accession | GSM1071027
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1071nnn/GSM1071027/suppl/GSM1071027_VFP-0129-6.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1071028 | GPL201 |
|
VFP-0142_G0_DF [HG-Focus]
|
VFP_G0_DF
|
patient no.: 142
Stage: G0
infecting serotype: 2
phase: Early Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 0
scan date: 2008-01-16
|
|
| Sample_geo_accession | GSM1071028
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1071nnn/GSM1071028/suppl/GSM1071028_VFP-0142-1.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1071029 | GPL201 |
|
VFP-0142_G3_DF [HG-Focus]
|
VFP_G3_DF
|
patient no.: 142
Stage: G3
infecting serotype: 2
phase: Early Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df0
scan date: 2008-01-16
|
|
| Sample_geo_accession | GSM1071029
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1071nnn/GSM1071029/suppl/GSM1071029_VFP-0142-5.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1071030 | GPL201 |
|
VFP-0142_G7_DF [HG-Focus]
|
VFP_G7_DF
|
patient no.: 142
Stage: G7
infecting serotype: 2
phase: Convalenscent
severity: DF (dengue fever)
fever days or defervescent (df) days: c
scan date: 2008-01-16
|
|
| Sample_geo_accession | GSM1071030
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1071nnn/GSM1071030/suppl/GSM1071030_VFP-0142-6.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1071031 | GPL201 |
|
VFP-0144_G2_DF [HG-Focus]
|
VFP_G2_DF
|
patient no.: 144
Stage: G2
infecting serotype: 1
phase: Early Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 2
scan date: 2008-01-16
|
|
| Sample_geo_accession | GSM1071031
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1071nnn/GSM1071031/suppl/GSM1071031_VFP-0144-1.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1071032 | GPL201 |
|
VFP-0144_G5_DF [HG-Focus]
|
VFP_G5_DF
|
patient no.: 144
Stage: G5
infecting serotype: 1
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df1
scan date: 2008-01-16
|
|
| Sample_geo_accession | GSM1071032
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1071nnn/GSM1071032/suppl/GSM1071032_VFP-0144-5.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1071033 | GPL201 |
|
VFP-0144_G7_DF [HG-Focus]
|
VFP_G7_DF
|
patient no.: 144
Stage: G7
infecting serotype: 1
phase: Convalenscent
severity: DF (dengue fever)
fever days or defervescent (df) days: c
scan date: 2008-01-16
|
|
| Sample_geo_accession | GSM1071033
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1071nnn/GSM1071033/suppl/GSM1071033_VFP-0144-6.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1071034 | GPL201 |
|
VFP-0145_G2_DF [HG-Focus]
|
VFP_G2_DF
|
patient no.: 145
Stage: G2
infecting serotype: 1
phase: Early Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 2
scan date: 2008-01-16
|
|
| Sample_geo_accession | GSM1071034
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1071nnn/GSM1071034/suppl/GSM1071034_VFP-0145-1.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1071035 | GPL201 |
|
VFP-0145_G5_DF [HG-Focus]
|
VFP_G5_DF
|
patient no.: 145
Stage: G5
infecting serotype: 1
phase: Late Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: df2
scan date: 2008-01-16
|
|
| Sample_geo_accession | GSM1071035
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1071nnn/GSM1071035/suppl/GSM1071035_VFP-0145-5.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1071036 | GPL201 |
|
VFP-0145_G7_DF [HG-Focus]
|
VFP_G7_DF
|
patient no.: 145
Stage: G7
infecting serotype: 1
phase: Convalenscent
severity: DF (dengue fever)
fever days or defervescent (df) days: c
scan date: 2008-01-16
|
|
| Sample_geo_accession | GSM1071036
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1071nnn/GSM1071036/suppl/GSM1071036_VFP-0145-6.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1071037 | GPL201 |
|
VFP-0003_G7_DF [HG-Focus]
|
VFP_G7_DF
|
patient no.: 3
Stage: G7
infecting serotype: 1
phase: Convalenscent
severity: DF (dengue fever)
fever days or defervescent (df) days: c
scan date: 2008-01-24
|
|
| Sample_geo_accession | GSM1071037
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1071nnn/GSM1071037/suppl/GSM1071037_VFP-0003-6.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
| | |
|
GSM1071038 | GPL201 |
|
VFP-0029_G1_DF [HG-Focus]
|
VFP_G1_DF
|
patient no.: 29
Stage: G1
infecting serotype: 1
phase: Early Acute
severity: DF (dengue fever)
fever days or defervescent (df) days: 1
scan date: 2008-01-18
|
|
| Sample_geo_accession | GSM1071038
| | Sample_status | Public on Jan 26 2013
| | Sample_submission_date | Jan 25 2013
| | Sample_last_update_date | Jan 26 2013
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_growth_protocol_ch1 | Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-paque according to manufacturer's instruction
| | Sample_growth_protocol_ch1 | Cells were frozen in 10% dimethyl sulfoxide (DMSO) in -70 ͦC until use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | The DMSO-cryopreserved cells were resuspended in 1 ml of TRI Reagent (Molecular Research Center, Woodlands, TX). The mixture was vortexed for 15 sec and allowed to stand at room temperature for 10 min. The manufacturer’s instructions for the preparation of total RNA were followed.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Double-stranded cDNA was synthesized in two steps with the SuperScript Choice system (GIBCO-BRL, Rockville, MD) and the reverse transcription primer T7-(dt)24 (59-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24-39] (Genset, La Jolla, CA). First-strand synthesis was carried out in a 20-ml reaction. Approximately 5 mg of total RNA or 1.0 mg of poly(A) RNA was annealed to 7 mg of T7-(dt)24 primer at 70°C for 10 min. Reverse transcription was carried out at 37°C for 1 hr in a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol(DTT), dATP, dCTP, dGTP, and dTTP (500 mM each), and SuperScript II reverse transcriptase (20,000 to 30,000 U/ml) and the reaction was terminated by placing the tube on ice. Second strand synthesis was carried out in 150 ml incorporating the entire 20-ml first-strand reaction mixture and the 130-ml second strand reaction mixture for a final concentration of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 0.15 mM b-NAD1, dATP, dCTP, dGTP, and dTTP (250 mM each), 1.2 mM DTT, DNA ligase (65 U/ml), DNA polymerase I (250 U/ml), and RNase H (13 U/ml). The mixture was incubated at 16°C for 2 hr, whereupon 2 ml of T4 DNA polymerase at 5 U/ml was added and the incubation continued at 16°C for 5 min. To terminate the reaction, 10 ml of 0.5 M EDTA was added. The cDNA was purified with phenol–chloroform–isoamyl alcohol (24:23:1) saturated with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (Ambion, Austin, TX). The purified cDNA was precipitated with 5 M ammonium acetate and absolute ethanol at 220°C for 20 min. The pellet was resuspended in 7–9 ml of RNase-free water to achieve a final concentration of between 0.25 and 0.65 mg/ml.
| | Sample_label_protocol_ch1 | Synthesis of biotin-labeled cRNA was carried out by in vitro transcription using the MEGAscript T7 in vitro transcription kit (Ambion). In accordance with the manufacturer’s instructions, 0.4–1.0 mg of double-stranded cDNA was placed into a 20-ml reaction mix, at room temperature, containing Ambion 13 reaction buffer and enzyme mix (proprietary). The labeling mix consisted of 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP and 1.9 mM biotinylated UTP (Enzo Diagnostics, Farmingdale, NY), and 5.6 mM CTP and 1.9 mM biotinylated CTP (Enzo Diagnostics). The reaction was incubated at 37°C for 5 hr. The biotin-labeled cRNA was purified with RNeasy spin columns (Qiagen,Valencia, CA) according to the manufacturer’s protocol. The biotin-labeled cRNA was fragmented in a 40-ml reaction mixture containing 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, incubated at 94°C for 35 min, and then put on ice.
| | Sample_hyb_protocol | The biotin-labeled and fragmented cRNA was hybridized to the HUGeneFL GeneChip array (Affymetrix) or to the TEST 3 array as described previously. Briefly, a 220-ml hybridization solution consisting of 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pM control Oligo B2 (59-bioGTCAAGATGCTACCGTTCAG-39) (Affymetrix), control cRNA cocktail (Bio B [150 pM], Bio C [500 pM], Bio D [2.5 nM], and Cre X [10 nM]; American Type Tissue Collection [Manassas, VA] and Lofstrand Laboratories [Gaithersburg, MD]), herring sperm DNA (0.1 mg/ml), and fragmented labeled sample cRNA (0.05 mg/ml) was heated to 95°C for 35 min, cooled to 40°C, and clarified by centrifugation. Hybridization was at 42°C in a rotisserie hybridization oven (model 320; Affymetrix) at 60 rpm or 16 hr. Following hybridization, the gene chip arrays were washed 10 times at 25°C with 63 SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4) and 0.005% Triton X-100 (pH 7.6), using the automated fluidics station protocol. Gene chip arrays were incubated at 50°C in 0.53 SSPE-T and 0.005% Triton X-100 for 20 min at 60 rpm in the rotisserie oven. Gene chip arrays were stained for 15 min at room temperature and at 60 rpm, with streptavidin–phycoerythrin (Molecular probes, Eugene, OR) stain solution at a final concentration of 10 mg/ml in 63SSPE-T buffer and acetylated bovine serum albumin (1.0 mg/ml; Sigma).
| | Sample_scan_protocol | Gene chip arrays were washed twice at room temperature with 63 SSPE-T buffer. Gene chip arrays were scanned with an HP GeneArray scanner (Hewlett-Packard, Santa Clara, CA) controlled by GeneChip 3.1 software (Affymetrix).
| | Sample_data_processing | Gene chips with a scaling factor >50 on dChip software (2005 version) were eliminated from further analysis [PMID 12803996 and 20078211]. Gene chips were also examined by QA/QC functions built in the Partek software according to the company’s User’s Manual. Affymetrix CEL files were imported to Partek Genomics Suite Version 6.6 (Partek). Gene chips that failed by QC metrics and appeared as outliers on Principal Component Analysis (PCA) were eliminated for further analysis. CEL files were normalized at the probe level using the Robust Multi-chip Average (RMA) method built into in Partek Genomic Suite software. RMA is a method of normalization of microarray data that leverages gene expression assessments made on Affymetrix gene chips. For each probeset, the technique generates an estimated value for the probe and chip effects that result in the overall pattern of probe set values observed in the data set. RMA is comprised of 3 calculation applications that address background correction, normalization of quantiles, and median polish summarization. Gene expression data were expressed as log2 values. Data sets from the two gene chip platforms were first normalized individually and further analyzed independently.
| | Sample_data_processing | Differentially expressed genes were analyzed using analysis of variance (ANOVA) built in the Partek software. Multi-way ANOVA was chosen based on the number of factors contributing to chip data variation. The following factors were taken in consideration as variation factors: subject #, scan date (date the gene chip experiment was done and the chip image was taken), infecting serotype, illness day (days of illness when sample was taken), and disease severity (samples associated with DF or DHF category). Genes considered being significant only by chance were defined by the false discovery rate (FDR≥5%) and were excluded from further analysis. In this study, genes with fold change of >2 or <-2 and a p value plus a FDR < 5% were counted to be significant. Heatmap and principal component analysis were used to visualize the most informative trends by showing the predominant gradients in the data set.
| | Sample_platform_id | GPL201
| | Sample_contact_name | peifang,,sun
| | Sample_contact_email | peifang.sun@med.navy.mil
| | Sample_contact_institute | Naval Medical Research Center
| | Sample_contact_address | 503 robert grant ave
| | Sample_contact_city | silver spring
| | Sample_contact_zip/postal_code | 20910
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1071nnn/GSM1071038/suppl/GSM1071038_VFP-0029-1.CEL.gz
| | Sample_series_id | GSE43777
| | Sample_data_row_count | 8793
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