Search results for the GEO ID: GSE13292  |
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|
GSM ID | GPL ID |
Select for analysis |
Title |
Source name |
Description |
Characteristics |
GSM335415 | GPL201 |
|
A_fresh_Method 1
|
Whole blood, fresh, no depletion_Affymetrix
|
Gender: M
Age: 31, Normal
Biosource: Peripheral whole blood
Donor ID: Donor A
Sample storage state: fresh
Sample protocol: Whole blood was incubated in a PAXGene tube (PreAnalytiX, a Qiagen BD Company, Valencia, CA, USA) for two hours and then RNA was isolated from the fresh sample.
Treatment protocol: Affymetrix one-cycle cDNA synthesis/Affymetrix IVT (no depletion_Affymetrix): No modifications of sample made prior to target synthesis.
Label protocol: Affymetrix One-Cycle: Target synthesis was performed following the Affymetrix GeneChip Expression Analysis Technical Manual, rev. 5 (http://www.affymetrix.com/support/technical/manual/expression_manual.affx). Using 1 µg of total RNA as input, messenger RNA was amplified and labeled in two steps. In the first step, mRNA was converted to double-stranded cDNA using Superscript Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) and an oligo-dT primer linked to a T7 RNA polymerase binding site sequence (Affymetrix, Santa Clara, CA, USA). Double-stranded cDNA was purified by phenol-chloroform-isoamyl alcohol extraction and a standard ethanol precipitation. In the second step, amplified and labeled cRNA (the target) was produced by a transcription reaction using T7 RNA polymerase and biotin-CTP (Affymetrix). Unincorporated nucleotides were removed using the RNeasy Mini kit (Qiagen) followed by an ethanol precipitation of the labeled target.
Target molecule: cRNA
Hyb protocol: Labeled cRNA targets were chemically fragmented at 95C for 35 min as per Affymetrix’s recommendations. The fragmented material was combined with Affymetrix hybridization controls: biotinylated control oligomer (B2) and biotinylated control cRNAs for BioB, BioC, BioD and CreX (Affymetrix) in hybridization buffer. Following the manufacturer’s recommendations, 6.5 µg of target were hybridized with the GeneChip Human Genome Focus array (Affymetrix) for 16 hours at 45C. Post-hybridization array processing was performed on the Fluidics Station 450 (Affymetrix); array staining and signal amplification were performed according to manufacturer’s recommendations.
|
01A-11H1H_Aaffy_270JR
|
| Sample_geo_accession | GSM335415
| | Sample_status | Public on Jan 30 2009
| | Sample_submission_date | Oct 21 2008
| | Sample_last_update_date | Jan 30 2009
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Unfractionated whole blood collection and RNA isolation were performed using the PAXgene Blood RNA Isolation System (PreAnalytiX, a Qiagen BD Company, Valencia, CA, USA). With the approval of the OHSU Institutional Review Board, blood was collected from healthy donors using eight PAXGene tubes per individual (donor). Four tubes per donor were frozen for subsequent processing and four tubes per donor were processed fresh (see individual sample annotation for storage state details). Following RNA isolation, DNase-treatment was performed as per PAXGene manufacturer’s recommendation. Samples were then pooled within donor and within RNA storage state (fresh/frozen) resulting in two tubes per donor (fresh/frozen). RNA recovery and quality was then assessed by examining UV 260/280 absorbance ratios and RNA size distribution on RNA Nano LabChips (Agilent Technologies, Santa Clara, CA, USA) processed on the Agilent 2100 Bioanalyzer. For inclusion in this study, the RNA mass per donor had to be greater than 12 µg for both fresh and frozen blood aliquots. Samples from three of the four donors (Donors A, C, D) met the quantitative and qualitative criteria and were used in the microarray analysis.
| | Sample_growth_protocol_ch1 | n/a
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | [see characteristics field for details]
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Amplifications and labelings were performed for each donor using one of the following four methods with RNA extracted from fresh and frozen blood samples: Method 1 - Affymetrix one-cycle cDNA synthesis/Affymetrix in vitro-transcription (IVT) on whole blood RNA; Method 2 - Affymetrix one-cycle cDNA/Affymetrix IVT with globin peptide nucleic acids (PNAs) inclusion during cDNA synthesis; and Method 3- RNA pre-treated with Ambion GLOBINclear beads to reduce globin transcripts prior to labeling with the Affymetrix one-cycle cDNA synthesis/Affymetrix IVT. In Method 4- cDNA target was synthesized from whole blood RNA using the NuGEN Ovation Biotin RNA Amplification and Labeling System. See individual sample annotation for protocol details.
| | Sample_hyb_protocol | [see characteristics field for details]
| | Sample_scan_protocol | The distribution of fluorescent material on the processed array was determined using the Affymetrix 3000 GeneArray laser scanner with the 7G upgrade. Image inspection was performed manually immediately following each scan. The array image scan was processed with Affymetrix Microarray Suite, version 5.0, (MAS 5.0) software.
| | Sample_data_processing | Low-level analysis (background correction, normalization, and gene summarization) of microarray data was performed with both Affymetrix Microarray Suite 5.0 (MAS 5.0) and Robust Multi-Array Average (RMA). Individual arrays were analyzed and scaled with MAS 5.0 using manufacturer’s default thresholds for detection calls and with target scaling factor set as 275. For RMA analysis, arrays were normalized in four separate groups based on target labeling method using RMA implemented in Affymetrix Expression Console (version 1.1).
| | Sample_platform_id | GPL201
| | Sample_contact_name | Christina,A,Harrington
| | Sample_contact_email | harringc@ohsu.edu
| | Sample_contact_phone | 503-418-2737
| | Sample_contact_fax | 503-418-9381
| | Sample_contact_laboratory | Affymetrix Microarray Core Facility
| | Sample_contact_department | Gene Microarray Shared Resource
| | Sample_contact_institute | Oregon Health and Science University
| | Sample_contact_address | 3303 SW Bond Ave
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM335nnn/GSM335415/suppl/GSM335415.CEL.gz
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM335nnn/GSM335415/suppl/GSM335415.CHP.gz
| | Sample_series_id | GSE13292
| | Sample_data_row_count | 8793
| | |
|
GSM335416 | GPL201 |
|
C_fresh_Method 1
|
Whole blood, fresh, no depletion_Affymetrix
|
Gender: M
Age: 25, Normal
Biosource: Peripheral whole blood
Donor ID: Donor C
Sample storage state: fresh
Sample protocol: Whole blood was incubated in a PAXGene tube (PreAnalytiX, a Qiagen BD Company, Valencia, CA, USA) for two hours and then RNA was isolated from the fresh sample.
Treatment protocol: Affymetrix one-cycle cDNA synthesis/Affymetrix IVT (no depletion_Affymetrix): No modifications of sample made prior to target synthesis.
Label protocol: Affymetrix One-Cycle: Target synthesis was performed following the Affymetrix GeneChip Expression Analysis Technical Manual, rev. 5 (http://www.affymetrix.com/support/technical/manual/expression_manual.affx). Using 1 µg of total RNA as input, messenger RNA was amplified and labeled in two steps. In the first step, mRNA was converted to double-stranded cDNA using Superscript Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) and an oligo-dT primer linked to a T7 RNA polymerase binding site sequence (Affymetrix, Santa Clara, CA, USA). Double-stranded cDNA was purified by phenol-chloroform-isoamyl alcohol extraction and a standard ethanol precipitation. In the second step, amplified and labeled cRNA (the target) was produced by a transcription reaction using T7 RNA polymerase and biotin-CTP (Affymetrix). Unincorporated nucleotides were removed using the RNeasy Mini kit (Qiagen) followed by an ethanol precipitation of the labeled target.
Target molecule: cRNA
Hyb protocol: Labeled cRNA targets were chemically fragmented at 95C for 35 min as per Affymetrix’s recommendations. The fragmented material was combined with Affymetrix hybridization controls: biotinylated control oligomer (B2) and biotinylated control cRNAs for BioB, BioC, BioD and CreX (Affymetrix) in hybridization buffer. Following the manufacturer’s recommendations, 6.5 µg of target were hybridized with the GeneChip Human Genome Focus array (Affymetrix) for 16 hours at 45C. Post-hybridization array processing was performed on the Fluidics Station 450 (Affymetrix); array staining and signal amplification were performed according to manufacturer’s recommendations.
|
03A-11H1H_Caffy_270JR
|
| Sample_geo_accession | GSM335416
| | Sample_status | Public on Jan 30 2009
| | Sample_submission_date | Oct 21 2008
| | Sample_last_update_date | Jan 30 2009
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Unfractionated whole blood collection and RNA isolation were performed using the PAXgene Blood RNA Isolation System (PreAnalytiX, a Qiagen BD Company, Valencia, CA, USA). With the approval of the OHSU Institutional Review Board, blood was collected from healthy donors using eight PAXGene tubes per individual (donor). Four tubes per donor were frozen for subsequent processing and four tubes per donor were processed fresh (see individual sample annotation for storage state details). Following RNA isolation, DNase-treatment was performed as per PAXGene manufacturer’s recommendation. Samples were then pooled within donor and within RNA storage state (fresh/frozen) resulting in two tubes per donor (fresh/frozen). RNA recovery and quality was then assessed by examining UV 260/280 absorbance ratios and RNA size distribution on RNA Nano LabChips (Agilent Technologies, Santa Clara, CA, USA) processed on the Agilent 2100 Bioanalyzer. For inclusion in this study, the RNA mass per donor had to be greater than 12 µg for both fresh and frozen blood aliquots. Samples from three of the four donors (Donors A, C, D) met the quantitative and qualitative criteria and were used in the microarray analysis.
| | Sample_growth_protocol_ch1 | n/a
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | [see characteristics field for details]
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Amplifications and labelings were performed for each donor using one of the following four methods with RNA extracted from fresh and frozen blood samples: Method 1 - Affymetrix one-cycle cDNA synthesis/Affymetrix in vitro-transcription (IVT) on whole blood RNA; Method 2 - Affymetrix one-cycle cDNA/Affymetrix IVT with globin peptide nucleic acids (PNAs) inclusion during cDNA synthesis; and Method 3- RNA pre-treated with Ambion GLOBINclear beads to reduce globin transcripts prior to labeling with the Affymetrix one-cycle cDNA synthesis/Affymetrix IVT. In Method 4- cDNA target was synthesized from whole blood RNA using the NuGEN Ovation Biotin RNA Amplification and Labeling System. See individual sample annotation for protocol details.
| | Sample_hyb_protocol | [see characteristics field for details]
| | Sample_scan_protocol | The distribution of fluorescent material on the processed array was determined using the Affymetrix 3000 GeneArray laser scanner with the 7G upgrade. Image inspection was performed manually immediately following each scan. The array image scan was processed with Affymetrix Microarray Suite, version 5.0, (MAS 5.0) software.
| | Sample_data_processing | Low-level analysis (background correction, normalization, and gene summarization) of microarray data was performed with both Affymetrix Microarray Suite 5.0 (MAS 5.0) and Robust Multi-Array Average (RMA). Individual arrays were analyzed and scaled with MAS 5.0 using manufacturer’s default thresholds for detection calls and with target scaling factor set as 275. For RMA analysis, arrays were normalized in four separate groups based on target labeling method using RMA implemented in Affymetrix Expression Console (version 1.1).
| | Sample_platform_id | GPL201
| | Sample_contact_name | Christina,A,Harrington
| | Sample_contact_email | harringc@ohsu.edu
| | Sample_contact_phone | 503-418-2737
| | Sample_contact_fax | 503-418-9381
| | Sample_contact_laboratory | Affymetrix Microarray Core Facility
| | Sample_contact_department | Gene Microarray Shared Resource
| | Sample_contact_institute | Oregon Health and Science University
| | Sample_contact_address | 3303 SW Bond Ave
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM335nnn/GSM335416/suppl/GSM335416.CEL.gz
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM335nnn/GSM335416/suppl/GSM335416.CHP.gz
| | Sample_series_id | GSE13292
| | Sample_data_row_count | 8793
| | |
|
GSM335417 | GPL201 |
|
D_fresh_Method 1
|
Whole blood, fresh, no depletion_Affymetrix
|
Gender: M
Age: 24, Normal
Biosource: Peripheral whole blood
Donor ID: Donor D
Sample storage state: fresh
Sample protocol: Whole blood was incubated in a PAXGene tube (PreAnalytiX, a Qiagen BD Company, Valencia, CA, USA) for two hours and then RNA was isolated from the fresh sample.
Treatment protocol: Affymetrix one-cycle cDNA synthesis/Affymetrix IVT (no depletion_Affymetrix): No modifications of sample made prior to target synthesis.
Label protocol: Affymetrix One-Cycle: Target synthesis was performed following the Affymetrix GeneChip Expression Analysis Technical Manual, rev. 5 (http://www.affymetrix.com/support/technical/manual/expression_manual.affx). Using 1 µg of total RNA as input, messenger RNA was amplified and labeled in two steps. In the first step, mRNA was converted to double-stranded cDNA using Superscript Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) and an oligo-dT primer linked to a T7 RNA polymerase binding site sequence (Affymetrix, Santa Clara, CA, USA). Double-stranded cDNA was purified by phenol-chloroform-isoamyl alcohol extraction and a standard ethanol precipitation. In the second step, amplified and labeled cRNA (the target) was produced by a transcription reaction using T7 RNA polymerase and biotin-CTP (Affymetrix). Unincorporated nucleotides were removed using the RNeasy Mini kit (Qiagen) followed by an ethanol precipitation of the labeled target.
Target molecule: cRNA
Hyb protocol: Labeled cRNA targets were chemically fragmented at 95C for 35 min as per Affymetrix’s recommendations. The fragmented material was combined with Affymetrix hybridization controls: biotinylated control oligomer (B2) and biotinylated control cRNAs for BioB, BioC, BioD and CreX (Affymetrix) in hybridization buffer. Following the manufacturer’s recommendations, 6.5 µg of target were hybridized with the GeneChip Human Genome Focus array (Affymetrix) for 16 hours at 45C. Post-hybridization array processing was performed on the Fluidics Station 450 (Affymetrix); array staining and signal amplification were performed according to manufacturer’s recommendations.
|
05A-11H1H_Daffy_270JR
|
| Sample_geo_accession | GSM335417
| | Sample_status | Public on Jan 30 2009
| | Sample_submission_date | Oct 21 2008
| | Sample_last_update_date | Jan 30 2009
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Unfractionated whole blood collection and RNA isolation were performed using the PAXgene Blood RNA Isolation System (PreAnalytiX, a Qiagen BD Company, Valencia, CA, USA). With the approval of the OHSU Institutional Review Board, blood was collected from healthy donors using eight PAXGene tubes per individual (donor). Four tubes per donor were frozen for subsequent processing and four tubes per donor were processed fresh (see individual sample annotation for storage state details). Following RNA isolation, DNase-treatment was performed as per PAXGene manufacturer’s recommendation. Samples were then pooled within donor and within RNA storage state (fresh/frozen) resulting in two tubes per donor (fresh/frozen). RNA recovery and quality was then assessed by examining UV 260/280 absorbance ratios and RNA size distribution on RNA Nano LabChips (Agilent Technologies, Santa Clara, CA, USA) processed on the Agilent 2100 Bioanalyzer. For inclusion in this study, the RNA mass per donor had to be greater than 12 µg for both fresh and frozen blood aliquots. Samples from three of the four donors (Donors A, C, D) met the quantitative and qualitative criteria and were used in the microarray analysis.
| | Sample_growth_protocol_ch1 | n/a
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | [see characteristics field for details]
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Amplifications and labelings were performed for each donor using one of the following four methods with RNA extracted from fresh and frozen blood samples: Method 1 - Affymetrix one-cycle cDNA synthesis/Affymetrix in vitro-transcription (IVT) on whole blood RNA; Method 2 - Affymetrix one-cycle cDNA/Affymetrix IVT with globin peptide nucleic acids (PNAs) inclusion during cDNA synthesis; and Method 3- RNA pre-treated with Ambion GLOBINclear beads to reduce globin transcripts prior to labeling with the Affymetrix one-cycle cDNA synthesis/Affymetrix IVT. In Method 4- cDNA target was synthesized from whole blood RNA using the NuGEN Ovation Biotin RNA Amplification and Labeling System. See individual sample annotation for protocol details.
| | Sample_hyb_protocol | [see characteristics field for details]
| | Sample_scan_protocol | The distribution of fluorescent material on the processed array was determined using the Affymetrix 3000 GeneArray laser scanner with the 7G upgrade. Image inspection was performed manually immediately following each scan. The array image scan was processed with Affymetrix Microarray Suite, version 5.0, (MAS 5.0) software.
| | Sample_data_processing | Low-level analysis (background correction, normalization, and gene summarization) of microarray data was performed with both Affymetrix Microarray Suite 5.0 (MAS 5.0) and Robust Multi-Array Average (RMA). Individual arrays were analyzed and scaled with MAS 5.0 using manufacturer’s default thresholds for detection calls and with target scaling factor set as 275. For RMA analysis, arrays were normalized in four separate groups based on target labeling method using RMA implemented in Affymetrix Expression Console (version 1.1).
| | Sample_platform_id | GPL201
| | Sample_contact_name | Christina,A,Harrington
| | Sample_contact_email | harringc@ohsu.edu
| | Sample_contact_phone | 503-418-2737
| | Sample_contact_fax | 503-418-9381
| | Sample_contact_laboratory | Affymetrix Microarray Core Facility
| | Sample_contact_department | Gene Microarray Shared Resource
| | Sample_contact_institute | Oregon Health and Science University
| | Sample_contact_address | 3303 SW Bond Ave
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM335nnn/GSM335417/suppl/GSM335417.CEL.gz
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM335nnn/GSM335417/suppl/GSM335417.CHP.gz
| | Sample_series_id | GSE13292
| | Sample_data_row_count | 8793
| | |
|
GSM335418 | GPL201 |
|
A_frozen_Method 1
|
Whole blood, frozen, no depletion_Affymetrix
|
Gender: M
Age: 31, Normal
Biosource: Peripheral whole blood
Donor ID: Donor A
Sample storage state: frozen
Sample protocol: Whole blood was incubated in a PAXGene tube (PreAnalytiX, a Qiagen BD Company, Valencia, CA, USA) for two hours and then transferred to a -80C freezer. RNA was isolated from the frozen PAXGene tube after storage at -80C for at least one week. Treatment protocol: Affymetrix one-cycle cDNA synthesis/Affymetrix IVT (no depletion_Affymetrix): No modifications of sample made prior to target synthesis.
Label protocol: Affymetrix One-Cycle: Target synthesis was performed following the Affymetrix GeneChip Expression Analysis Technical Manual, rev. 5 (http://www.affymetrix.com/support/technical/manual/expression_manual.affx). Using 1 µg of total RNA as input, messenger RNA was amplified and labeled in two steps. In the first step, mRNA was converted to double-stranded cDNA using Superscript Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) and an oligo-dT primer linked to a T7 RNA polymerase binding site sequence (Affymetrix, Santa Clara, CA, USA). Double-stranded cDNA was purified by phenol-chloroform-isoamyl alcohol extraction and a standard ethanol precipitation. In the second step, amplified and labeled cRNA (the target) was produced by a transcription reaction using T7 RNA polymerase and biotin-CTP (Affymetrix). Unincorporated nucleotides were removed using the RNeasy Mini kit (Qiagen) followed by an ethanol precipitation of the labeled target.
Target molecule: cRNA
Hyb protocol: Labeled cRNA targets were chemically fragmented at 95C for 35 min as per Affymetrix’s recommendations. The fragmented material was combined with Affymetrix hybridization controls: biotinylated control oligomer (B2) and biotinylated control cRNAs for BioB, BioC, BioD and CreX (Affymetrix) in hybridization buffer. Following the manufacturer’s recommendations, 6.5 µg of target were hybridized with the GeneChip Human Genome Focus array (Affymetrix) for 16 hours at 45C. Post-hybridization array processing was performed on the Fluidics Station 450 (Affymetrix); array staining and signal amplification were performed according to manufacturer’s recommendations.
|
07A-11H1H_Afaffy_270JR
|
| Sample_geo_accession | GSM335418
| | Sample_status | Public on Jan 30 2009
| | Sample_submission_date | Oct 21 2008
| | Sample_last_update_date | Jan 30 2009
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Unfractionated whole blood collection and RNA isolation were performed using the PAXgene Blood RNA Isolation System (PreAnalytiX, a Qiagen BD Company, Valencia, CA, USA). With the approval of the OHSU Institutional Review Board, blood was collected from healthy donors using eight PAXGene tubes per individual (donor). Four tubes per donor were frozen for subsequent processing and four tubes per donor were processed fresh (see individual sample annotation for storage state details). Following RNA isolation, DNase-treatment was performed as per PAXGene manufacturer’s recommendation. Samples were then pooled within donor and within RNA storage state (fresh/frozen) resulting in two tubes per donor (fresh/frozen). RNA recovery and quality was then assessed by examining UV 260/280 absorbance ratios and RNA size distribution on RNA Nano LabChips (Agilent Technologies, Santa Clara, CA, USA) processed on the Agilent 2100 Bioanalyzer. For inclusion in this study, the RNA mass per donor had to be greater than 12 µg for both fresh and frozen blood aliquots. Samples from three of the four donors (Donors A, C, D) met the quantitative and qualitative criteria and were used in the microarray analysis.
| | Sample_growth_protocol_ch1 | n/a
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | [see characteristics field for details]
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Amplifications and labelings were performed for each donor using one of the following four methods with RNA extracted from fresh and frozen blood samples: Method 1 - Affymetrix one-cycle cDNA synthesis/Affymetrix in vitro-transcription (IVT) on whole blood RNA; Method 2 - Affymetrix one-cycle cDNA/Affymetrix IVT with globin peptide nucleic acids (PNAs) inclusion during cDNA synthesis; and Method 3- RNA pre-treated with Ambion GLOBINclear beads to reduce globin transcripts prior to labeling with the Affymetrix one-cycle cDNA synthesis/Affymetrix IVT. In Method 4- cDNA target was synthesized from whole blood RNA using the NuGEN Ovation Biotin RNA Amplification and Labeling System. See individual sample annotation for protocol details.
| | Sample_hyb_protocol | [see characteristics field for details]
| | Sample_scan_protocol | The distribution of fluorescent material on the processed array was determined using the Affymetrix 3000 GeneArray laser scanner with the 7G upgrade. Image inspection was performed manually immediately following each scan. The array image scan was processed with Affymetrix Microarray Suite, version 5.0, (MAS 5.0) software.
| | Sample_data_processing | Low-level analysis (background correction, normalization, and gene summarization) of microarray data was performed with both Affymetrix Microarray Suite 5.0 (MAS 5.0) and Robust Multi-Array Average (RMA). Individual arrays were analyzed and scaled with MAS 5.0 using manufacturer’s default thresholds for detection calls and with target scaling factor set as 275. For RMA analysis, arrays were normalized in four separate groups based on target labeling method using RMA implemented in Affymetrix Expression Console (version 1.1).
| | Sample_platform_id | GPL201
| | Sample_contact_name | Christina,A,Harrington
| | Sample_contact_email | harringc@ohsu.edu
| | Sample_contact_phone | 503-418-2737
| | Sample_contact_fax | 503-418-9381
| | Sample_contact_laboratory | Affymetrix Microarray Core Facility
| | Sample_contact_department | Gene Microarray Shared Resource
| | Sample_contact_institute | Oregon Health and Science University
| | Sample_contact_address | 3303 SW Bond Ave
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM335nnn/GSM335418/suppl/GSM335418.CEL.gz
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM335nnn/GSM335418/suppl/GSM335418.CHP.gz
| | Sample_series_id | GSE13292
| | Sample_data_row_count | 8793
| | |
|
GSM335419 | GPL201 |
|
C_frozen_Method 1
|
Whole blood, frozen, no depletion_Affymetrix
|
Gender: M
Age: 25, Normal
Biosource: Peripheral whole blood
Donor ID: Donor C
Sample storage state: frozen
Sample protocol: Whole blood was incubated in a PAXGene tube (PreAnalytiX, a Qiagen BD Company, Valencia, CA, USA) for two hours and then transferred to a -80C freezer. RNA was isolated from the frozen PAXGene tube after storage at -80C for at least one week. Treatment protocol: Affymetrix one-cycle cDNA synthesis/Affymetrix IVT (no depletion_Affymetrix): No modifications of sample made prior to target synthesis.
Label protocol: Affymetrix One-Cycle: Target synthesis was performed following the Affymetrix GeneChip Expression Analysis Technical Manual, rev. 5 (http://www.affymetrix.com/support/technical/manual/expression_manual.affx). Using 1 µg of total RNA as input, messenger RNA was amplified and labeled in two steps. In the first step, mRNA was converted to double-stranded cDNA using Superscript Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) and an oligo-dT primer linked to a T7 RNA polymerase binding site sequence (Affymetrix, Santa Clara, CA, USA). Double-stranded cDNA was purified by phenol-chloroform-isoamyl alcohol extraction and a standard ethanol precipitation. In the second step, amplified and labeled cRNA (the target) was produced by a transcription reaction using T7 RNA polymerase and biotin-CTP (Affymetrix). Unincorporated nucleotides were removed using the RNeasy Mini kit (Qiagen) followed by an ethanol precipitation of the labeled target.
Target molecule: cRNA
Hyb protocol: Labeled cRNA targets were chemically fragmented at 95C for 35 min as per Affymetrix’s recommendations. The fragmented material was combined with Affymetrix hybridization controls: biotinylated control oligomer (B2) and biotinylated control cRNAs for BioB, BioC, BioD and CreX (Affymetrix) in hybridization buffer. Following the manufacturer’s recommendations, 6.5 µg of target were hybridized with the GeneChip Human Genome Focus array (Affymetrix) for 16 hours at 45C. Post-hybridization array processing was performed on the Fluidics Station 450 (Affymetrix); array staining and signal amplification were performed according to manufacturer’s recommendations.
|
09A-11H1H_Cfaffy_270JR
|
| Sample_geo_accession | GSM335419
| | Sample_status | Public on Jan 30 2009
| | Sample_submission_date | Oct 21 2008
| | Sample_last_update_date | Jan 30 2009
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Unfractionated whole blood collection and RNA isolation were performed using the PAXgene Blood RNA Isolation System (PreAnalytiX, a Qiagen BD Company, Valencia, CA, USA). With the approval of the OHSU Institutional Review Board, blood was collected from healthy donors using eight PAXGene tubes per individual (donor). Four tubes per donor were frozen for subsequent processing and four tubes per donor were processed fresh (see individual sample annotation for storage state details). Following RNA isolation, DNase-treatment was performed as per PAXGene manufacturer’s recommendation. Samples were then pooled within donor and within RNA storage state (fresh/frozen) resulting in two tubes per donor (fresh/frozen). RNA recovery and quality was then assessed by examining UV 260/280 absorbance ratios and RNA size distribution on RNA Nano LabChips (Agilent Technologies, Santa Clara, CA, USA) processed on the Agilent 2100 Bioanalyzer. For inclusion in this study, the RNA mass per donor had to be greater than 12 µg for both fresh and frozen blood aliquots. Samples from three of the four donors (Donors A, C, D) met the quantitative and qualitative criteria and were used in the microarray analysis.
| | Sample_growth_protocol_ch1 | n/a
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | [see characteristics field for details]
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Amplifications and labelings were performed for each donor using one of the following four methods with RNA extracted from fresh and frozen blood samples: Method 1 - Affymetrix one-cycle cDNA synthesis/Affymetrix in vitro-transcription (IVT) on whole blood RNA; Method 2 - Affymetrix one-cycle cDNA/Affymetrix IVT with globin peptide nucleic acids (PNAs) inclusion during cDNA synthesis; and Method 3- RNA pre-treated with Ambion GLOBINclear beads to reduce globin transcripts prior to labeling with the Affymetrix one-cycle cDNA synthesis/Affymetrix IVT. In Method 4- cDNA target was synthesized from whole blood RNA using the NuGEN Ovation Biotin RNA Amplification and Labeling System. See individual sample annotation for protocol details.
| | Sample_hyb_protocol | [see characteristics field for details]
| | Sample_scan_protocol | The distribution of fluorescent material on the processed array was determined using the Affymetrix 3000 GeneArray laser scanner with the 7G upgrade. Image inspection was performed manually immediately following each scan. The array image scan was processed with Affymetrix Microarray Suite, version 5.0, (MAS 5.0) software.
| | Sample_data_processing | Low-level analysis (background correction, normalization, and gene summarization) of microarray data was performed with both Affymetrix Microarray Suite 5.0 (MAS 5.0) and Robust Multi-Array Average (RMA). Individual arrays were analyzed and scaled with MAS 5.0 using manufacturer’s default thresholds for detection calls and with target scaling factor set as 275. For RMA analysis, arrays were normalized in four separate groups based on target labeling method using RMA implemented in Affymetrix Expression Console (version 1.1).
| | Sample_platform_id | GPL201
| | Sample_contact_name | Christina,A,Harrington
| | Sample_contact_email | harringc@ohsu.edu
| | Sample_contact_phone | 503-418-2737
| | Sample_contact_fax | 503-418-9381
| | Sample_contact_laboratory | Affymetrix Microarray Core Facility
| | Sample_contact_department | Gene Microarray Shared Resource
| | Sample_contact_institute | Oregon Health and Science University
| | Sample_contact_address | 3303 SW Bond Ave
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM335nnn/GSM335419/suppl/GSM335419.CEL.gz
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM335nnn/GSM335419/suppl/GSM335419.CHP.gz
| | Sample_series_id | GSE13292
| | Sample_data_row_count | 8793
| | |
|
GSM335420 | GPL201 |
|
D_frozen_Method 1
|
Whole blood, frozen, no depletion_Affymetrix
|
Gender: M
Age: 24, Normal
Biosource: Peripheral whole blood
Donor ID: Donor D
Sample storage state: frozen
Sample protocol: Whole blood was incubated in a PAXGene tube (PreAnalytiX, a Qiagen BD Company, Valencia, CA, USA) for two hours and then transferred to a -80C freezer. RNA was isolated from the frozen PAXGene tube after storage at -80C for at least one week. Treatment protocol: Affymetrix one-cycle cDNA synthesis/Affymetrix IVT (no depletion_Affymetrix): No modifications of sample made prior to target synthesis.
Label protocol: Affymetrix One-Cycle: Target synthesis was performed following the Affymetrix GeneChip Expression Analysis Technical Manual, rev. 5 (http://www.affymetrix.com/support/technical/manual/expression_manual.affx). Using 1 µg of total RNA as input, messenger RNA was amplified and labeled in two steps. In the first step, mRNA was converted to double-stranded cDNA using Superscript Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) and an oligo-dT primer linked to a T7 RNA polymerase binding site sequence (Affymetrix, Santa Clara, CA, USA). Double-stranded cDNA was purified by phenol-chloroform-isoamyl alcohol extraction and a standard ethanol precipitation. In the second step, amplified and labeled cRNA (the target) was produced by a transcription reaction using T7 RNA polymerase and biotin-CTP (Affymetrix). Unincorporated nucleotides were removed using the RNeasy Mini kit (Qiagen) followed by an ethanol precipitation of the labeled target.
Target molecule: cRNA
Hyb protocol: Labeled cRNA targets were chemically fragmented at 95C for 35 min as per Affymetrix’s recommendations. The fragmented material was combined with Affymetrix hybridization controls: biotinylated control oligomer (B2) and biotinylated control cRNAs for BioB, BioC, BioD and CreX (Affymetrix) in hybridization buffer. Following the manufacturer’s recommendations, 6.5 µg of target were hybridized with the GeneChip Human Genome Focus array (Affymetrix) for 16 hours at 45C. Post-hybridization array processing was performed on the Fluidics Station 450 (Affymetrix); array staining and signal amplification were performed according to manufacturer’s recommendations.
|
11A-11H1H_Dfaffy_270JR
|
| Sample_geo_accession | GSM335420
| | Sample_status | Public on Jan 30 2009
| | Sample_submission_date | Oct 21 2008
| | Sample_last_update_date | Jan 30 2009
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Unfractionated whole blood collection and RNA isolation were performed using the PAXgene Blood RNA Isolation System (PreAnalytiX, a Qiagen BD Company, Valencia, CA, USA). With the approval of the OHSU Institutional Review Board, blood was collected from healthy donors using eight PAXGene tubes per individual (donor). Four tubes per donor were frozen for subsequent processing and four tubes per donor were processed fresh (see individual sample annotation for storage state details). Following RNA isolation, DNase-treatment was performed as per PAXGene manufacturer’s recommendation. Samples were then pooled within donor and within RNA storage state (fresh/frozen) resulting in two tubes per donor (fresh/frozen). RNA recovery and quality was then assessed by examining UV 260/280 absorbance ratios and RNA size distribution on RNA Nano LabChips (Agilent Technologies, Santa Clara, CA, USA) processed on the Agilent 2100 Bioanalyzer. For inclusion in this study, the RNA mass per donor had to be greater than 12 µg for both fresh and frozen blood aliquots. Samples from three of the four donors (Donors A, C, D) met the quantitative and qualitative criteria and were used in the microarray analysis.
| | Sample_growth_protocol_ch1 | n/a
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | [see characteristics field for details]
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Amplifications and labelings were performed for each donor using one of the following four methods with RNA extracted from fresh and frozen blood samples: Method 1 - Affymetrix one-cycle cDNA synthesis/Affymetrix in vitro-transcription (IVT) on whole blood RNA; Method 2 - Affymetrix one-cycle cDNA/Affymetrix IVT with globin peptide nucleic acids (PNAs) inclusion during cDNA synthesis; and Method 3- RNA pre-treated with Ambion GLOBINclear beads to reduce globin transcripts prior to labeling with the Affymetrix one-cycle cDNA synthesis/Affymetrix IVT. In Method 4- cDNA target was synthesized from whole blood RNA using the NuGEN Ovation Biotin RNA Amplification and Labeling System. See individual sample annotation for protocol details.
| | Sample_hyb_protocol | [see characteristics field for details]
| | Sample_scan_protocol | The distribution of fluorescent material on the processed array was determined using the Affymetrix 3000 GeneArray laser scanner with the 7G upgrade. Image inspection was performed manually immediately following each scan. The array image scan was processed with Affymetrix Microarray Suite, version 5.0, (MAS 5.0) software.
| | Sample_data_processing | Low-level analysis (background correction, normalization, and gene summarization) of microarray data was performed with both Affymetrix Microarray Suite 5.0 (MAS 5.0) and Robust Multi-Array Average (RMA). Individual arrays were analyzed and scaled with MAS 5.0 using manufacturer’s default thresholds for detection calls and with target scaling factor set as 275. For RMA analysis, arrays were normalized in four separate groups based on target labeling method using RMA implemented in Affymetrix Expression Console (version 1.1).
| | Sample_platform_id | GPL201
| | Sample_contact_name | Christina,A,Harrington
| | Sample_contact_email | harringc@ohsu.edu
| | Sample_contact_phone | 503-418-2737
| | Sample_contact_fax | 503-418-9381
| | Sample_contact_laboratory | Affymetrix Microarray Core Facility
| | Sample_contact_department | Gene Microarray Shared Resource
| | Sample_contact_institute | Oregon Health and Science University
| | Sample_contact_address | 3303 SW Bond Ave
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM335nnn/GSM335420/suppl/GSM335420.CEL.gz
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM335nnn/GSM335420/suppl/GSM335420.CHP.gz
| | Sample_series_id | GSE13292
| | Sample_data_row_count | 8793
| | |
|
GSM335421 | GPL201 |
|
A_fresh_Method 2
|
Whole blood, fresh, Globin PNAs_Affymetrix
|
Gender: M
Age: 31, Normal
Biosource: Peripheral whole blood
Donor ID: Donor A
Sample storage state: fresh
Sample protocol: Whole blood was incubated in a PAXGene tube (PreAnalytiX, a Qiagen BD Company, Valencia, CA, USA) for two hours and then RNA was isolated from the fresh sample.
Treatment protocol: Affymetrix one-cycle cDNA synthesis/Affymetrix IVT with PNA treatment of total RNA (Globin PNAs_Affymetrix):
The standard Affymetrix amplification and labeling was performed with the following modification: four peptide nucleic acid (PNA) oligonucleotides (Applied Biosystems, Foster City, CA, USA) designed to anneal to human α- and β-globin mRNA were added to the total RNA preparations immediately prior to initiation of the target synthesis step in order to bind globin mRNA and block cDNA synthesis. The PNA oligonucleotide stocks were prepared on day of use at concentrations recommended by Affymetrix (www.affymetrix.com/support/technical/byproduct.affx?product=bloodrna). Globin reduction PNA master mix was prepared by adding equal amounts of the globin PNA stocks. Five µg of total RNA were incubated with oligo-dT primer and 1 µl PNA master mix for 10 minutes at 70C prior to first strand cDNA synthesis.
Label protocol: Affymetrix One-Cycle: Target synthesis was performed following the Affymetrix GeneChip Expression Analysis Technical Manual, rev. 5 (http://www.affymetrix.com/support/technical/manual/expression_manual.affx). Using 1 µg of total RNA as input, messenger RNA was amplified and labeled in two steps. In the first step, mRNA was converted to double-stranded cDNA using Superscript Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) and an oligo-dT primer linked to a T7 RNA polymerase binding site sequence (Affymetrix, Santa Clara, CA, USA). Double-stranded cDNA was purified by phenol-chloroform-isoamyl alcohol extraction and a standard ethanol precipitation. In the second step, amplified and labeled cRNA (the target) was produced by a transcription reaction using T7 RNA polymerase and biotin-CTP (Affymetrix). Unincorporated nucleotides were removed using the RNeasy Mini kit (Qiagen) followed by an ethanol precipitation of the labeled target.
Target molecule: cRNA
Hyb protocol: Labeled cRNA targets were chemically fragmented at 95C for 35 min as per Affymetrix’s recommendations. The fragmented material was combined with Affymetrix hybridization controls: biotinylated control oligomer (B2) and biotinylated control cRNAs for BioB, BioC, BioD and CreX (Affymetrix) in hybridization buffer. Following the manufacturer’s recommendations, 6.5 µg of target were hybridized with the GeneChip Human Genome Focus array (Affymetrix) for 16 hours at 45C. Post-hybridization array processing was performed on the Fluidics Station 450 (Affymetrix); array staining and signal amplification were performed according to manufacturer’s recommendations.
|
02A-11H1H_Apna_270JR
|
| Sample_geo_accession | GSM335421
| | Sample_status | Public on Jan 30 2009
| | Sample_submission_date | Oct 21 2008
| | Sample_last_update_date | Jan 30 2009
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Unfractionated whole blood collection and RNA isolation were performed using the PAXgene Blood RNA Isolation System (PreAnalytiX, a Qiagen BD Company, Valencia, CA, USA). With the approval of the OHSU Institutional Review Board, blood was collected from healthy donors using eight PAXGene tubes per individual (donor). Four tubes per donor were frozen for subsequent processing and four tubes per donor were processed fresh (see individual sample annotation for storage state details). Following RNA isolation, DNase-treatment was performed as per PAXGene manufacturer’s recommendation. Samples were then pooled within donor and within RNA storage state (fresh/frozen) resulting in two tubes per donor (fresh/frozen). RNA recovery and quality was then assessed by examining UV 260/280 absorbance ratios and RNA size distribution on RNA Nano LabChips (Agilent Technologies, Santa Clara, CA, USA) processed on the Agilent 2100 Bioanalyzer. For inclusion in this study, the RNA mass per donor had to be greater than 12 µg for both fresh and frozen blood aliquots. Samples from three of the four donors (Donors A, C, D) met the quantitative and qualitative criteria and were used in the microarray analysis.
| | Sample_growth_protocol_ch1 | n/a
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | [see characteristics field for details]
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Amplifications and labelings were performed for each donor using one of the following four methods with RNA extracted from fresh and frozen blood samples: Method 1 - Affymetrix one-cycle cDNA synthesis/Affymetrix in vitro-transcription (IVT) on whole blood RNA; Method 2 - Affymetrix one-cycle cDNA/Affymetrix IVT with globin peptide nucleic acids (PNAs) inclusion during cDNA synthesis; and Method 3- RNA pre-treated with Ambion GLOBINclear beads to reduce globin transcripts prior to labeling with the Affymetrix one-cycle cDNA synthesis/Affymetrix IVT. In Method 4- cDNA target was synthesized from whole blood RNA using the NuGEN Ovation Biotin RNA Amplification and Labeling System. See individual sample annotation for protocol details.
| | Sample_hyb_protocol | [see characteristics field for details]
| | Sample_scan_protocol | The distribution of fluorescent material on the processed array was determined using the Affymetrix 3000 GeneArray laser scanner with the 7G upgrade. Image inspection was performed manually immediately following each scan. The array image scan was processed with Affymetrix Microarray Suite, version 5.0, (MAS 5.0) software.
| | Sample_data_processing | Low-level analysis (background correction, normalization, and gene summarization) of microarray data was performed with both Affymetrix Microarray Suite 5.0 (MAS 5.0) and Robust Multi-Array Average (RMA). Individual arrays were analyzed and scaled with MAS 5.0 using manufacturer’s default thresholds for detection calls and with target scaling factor set as 275. For RMA analysis, arrays were normalized in four separate groups based on target labeling method using RMA implemented in Affymetrix Expression Console (version 1.1).
| | Sample_platform_id | GPL201
| | Sample_contact_name | Christina,A,Harrington
| | Sample_contact_email | harringc@ohsu.edu
| | Sample_contact_phone | 503-418-2737
| | Sample_contact_fax | 503-418-9381
| | Sample_contact_laboratory | Affymetrix Microarray Core Facility
| | Sample_contact_department | Gene Microarray Shared Resource
| | Sample_contact_institute | Oregon Health and Science University
| | Sample_contact_address | 3303 SW Bond Ave
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM335nnn/GSM335421/suppl/GSM335421.CEL.gz
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM335nnn/GSM335421/suppl/GSM335421.CHP.gz
| | Sample_series_id | GSE13292
| | Sample_data_row_count | 8793
| | |
|
GSM335422 | GPL201 |
|
C_fresh_Method 2
|
Whole blood, fresh, Globin PNAs_Affymetrix
|
Gender: M
Age: 25, Normal
Biosource: Peripheral whole blood
Donor ID: Donor C
Sample storage state: fresh
Sample protocol: Whole blood was incubated in a PAXGene tube (PreAnalytiX, a Qiagen BD Company, Valencia, CA, USA) for two hours and then RNA was isolated from the fresh sample.
Treatment protocol: Affymetrix one-cycle cDNA synthesis/Affymetrix IVT with PNA treatment of total RNA (Globin PNAs_Affymetrix): The standard Affymetrix amplification and labeling was performed with the following modification: four peptide nucleic acid (PNA) oligonucleotides (Applied Biosystems, Foster City, CA, USA) designed to anneal to human α- and β-globin mRNA were added to the total RNA preparations immediately prior to initiation of the target synthesis step in order to bind globin mRNA and block cDNA synthesis. The PNA oligonucleotide stocks were prepared on day of use at concentrations recommended by Affymetrix (www.affymetrix.com/support/technical/byproduct.affx?product=bloodrna). Globin reduction PNA master mix was prepared by adding equal amounts of the globin PNA stocks. Five µg of total RNA were incubated with oligo-dT primer and 1 µl PNA master mix for 10 minutes at 70C prior to first strand cDNA synthesis.
Label protocol: Affymetrix One-Cycle: Target synthesis was performed following the Affymetrix GeneChip Expression Analysis Technical Manual, rev. 5 (http://www.affymetrix.com/support/technical/manual/expression_manual.affx). Using 1 µg of total RNA as input, messenger RNA was amplified and labeled in two steps. In the first step, mRNA was converted to double-stranded cDNA using Superscript Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) and an oligo-dT primer linked to a T7 RNA polymerase binding site sequence (Affymetrix, Santa Clara, CA, USA). Double-stranded cDNA was purified by phenol-chloroform-isoamyl alcohol extraction and a standard ethanol precipitation. In the second step, amplified and labeled cRNA (the target) was produced by a transcription reaction using T7 RNA polymerase and biotin-CTP (Affymetrix). Unincorporated nucleotides were removed using the RNeasy Mini kit (Qiagen) followed by an ethanol precipitation of the labeled target.
Target molecule: cRNA
Hyb protocol: Labeled cRNA targets were chemically fragmented at 95C for 35 min as per Affymetrix’s recommendations. The fragmented material was combined with Affymetrix hybridization controls: biotinylated control oligomer (B2) and biotinylated control cRNAs for BioB, BioC, BioD and CreX (Affymetrix) in hybridization buffer. Following the manufacturer’s recommendations, 6.5 µg of target were hybridized with the GeneChip Human Genome Focus array (Affymetrix) for 16 hours at 45C. Post-hybridization array processing was performed on the Fluidics Station 450 (Affymetrix); array staining and signal amplification were performed according to manufacturer’s recommendations.
|
04A-11H1H_Cpna_270JR
|
| Sample_geo_accession | GSM335422
| | Sample_status | Public on Jan 30 2009
| | Sample_submission_date | Oct 21 2008
| | Sample_last_update_date | Jan 30 2009
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Unfractionated whole blood collection and RNA isolation were performed using the PAXgene Blood RNA Isolation System (PreAnalytiX, a Qiagen BD Company, Valencia, CA, USA). With the approval of the OHSU Institutional Review Board, blood was collected from healthy donors using eight PAXGene tubes per individual (donor). Four tubes per donor were frozen for subsequent processing and four tubes per donor were processed fresh (see individual sample annotation for storage state details). Following RNA isolation, DNase-treatment was performed as per PAXGene manufacturer’s recommendation. Samples were then pooled within donor and within RNA storage state (fresh/frozen) resulting in two tubes per donor (fresh/frozen). RNA recovery and quality was then assessed by examining UV 260/280 absorbance ratios and RNA size distribution on RNA Nano LabChips (Agilent Technologies, Santa Clara, CA, USA) processed on the Agilent 2100 Bioanalyzer. For inclusion in this study, the RNA mass per donor had to be greater than 12 µg for both fresh and frozen blood aliquots. Samples from three of the four donors (Donors A, C, D) met the quantitative and qualitative criteria and were used in the microarray analysis.
| | Sample_growth_protocol_ch1 | n/a
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | [see characteristics field for details]
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Amplifications and labelings were performed for each donor using one of the following four methods with RNA extracted from fresh and frozen blood samples: Method 1 - Affymetrix one-cycle cDNA synthesis/Affymetrix in vitro-transcription (IVT) on whole blood RNA; Method 2 - Affymetrix one-cycle cDNA/Affymetrix IVT with globin peptide nucleic acids (PNAs) inclusion during cDNA synthesis; and Method 3- RNA pre-treated with Ambion GLOBINclear beads to reduce globin transcripts prior to labeling with the Affymetrix one-cycle cDNA synthesis/Affymetrix IVT. In Method 4- cDNA target was synthesized from whole blood RNA using the NuGEN Ovation Biotin RNA Amplification and Labeling System. See individual sample annotation for protocol details.
| | Sample_hyb_protocol | [see characteristics field for details]
| | Sample_scan_protocol | The distribution of fluorescent material on the processed array was determined using the Affymetrix 3000 GeneArray laser scanner with the 7G upgrade. Image inspection was performed manually immediately following each scan. The array image scan was processed with Affymetrix Microarray Suite, version 5.0, (MAS 5.0) software.
| | Sample_data_processing | Low-level analysis (background correction, normalization, and gene summarization) of microarray data was performed with both Affymetrix Microarray Suite 5.0 (MAS 5.0) and Robust Multi-Array Average (RMA). Individual arrays were analyzed and scaled with MAS 5.0 using manufacturer’s default thresholds for detection calls and with target scaling factor set as 275. For RMA analysis, arrays were normalized in four separate groups based on target labeling method using RMA implemented in Affymetrix Expression Console (version 1.1).
| | Sample_platform_id | GPL201
| | Sample_contact_name | Christina,A,Harrington
| | Sample_contact_email | harringc@ohsu.edu
| | Sample_contact_phone | 503-418-2737
| | Sample_contact_fax | 503-418-9381
| | Sample_contact_laboratory | Affymetrix Microarray Core Facility
| | Sample_contact_department | Gene Microarray Shared Resource
| | Sample_contact_institute | Oregon Health and Science University
| | Sample_contact_address | 3303 SW Bond Ave
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM335nnn/GSM335422/suppl/GSM335422.CEL.gz
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM335nnn/GSM335422/suppl/GSM335422.CHP.gz
| | Sample_series_id | GSE13292
| | Sample_data_row_count | 8793
| | |
|
GSM335423 | GPL201 |
|
D_fresh_Method 2
|
Whole blood, fresh, Globin PNAs_Affymetrix
|
Gender: M
Age: 24, Normal
Biosource: Peripheral whole blood
Donor ID: Donor D
Sample storage state: fresh
Sample protocol: Whole blood was incubated in a PAXGene tube (PreAnalytiX, a Qiagen BD Company, Valencia, CA, USA) for two hours and then RNA was isolated from the fresh sample.
Treatment protocol: Affymetrix one-cycle cDNA synthesis/Affymetrix IVT with PNA treatment of total RNA (Globin PNAs_Affymetrix): The standard Affymetrix amplification and labeling was performed with the following modification: four peptide nucleic acid (PNA) oligonucleotides (Applied Biosystems, Foster City, CA, USA) designed to anneal to human α- and β-globin mRNA were added to the total RNA preparations immediately prior to initiation of the target synthesis step in order to bind globin mRNA and block cDNA synthesis. The PNA oligonucleotide stocks were prepared on day of use at concentrations recommended by Affymetrix (www.affymetrix.com/support/technical/byproduct.affx?product=bloodrna). Globin reduction PNA master mix was prepared by adding equal amounts of the globin PNA stocks. Five µg of total RNA were incubated with oligo-dT primer and 1 µl PNA master mix for 10 minutes at 70C prior to first strand cDNA synthesis.
Label protocol: Affymetrix One-Cycle: Target synthesis was performed following the Affymetrix GeneChip Expression Analysis Technical Manual, rev. 5 (http://www.affymetrix.com/support/technical/manual/expression_manual.affx). Using 1 µg of total RNA as input, messenger RNA was amplified and labeled in two steps. In the first step, mRNA was converted to double-stranded cDNA using Superscript Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) and an oligo-dT primer linked to a T7 RNA polymerase binding site sequence (Affymetrix, Santa Clara, CA, USA). Double-stranded cDNA was purified by phenol-chloroform-isoamyl alcohol extraction and a standard ethanol precipitation. In the second step, amplified and labeled cRNA (the target) was produced by a transcription reaction using T7 RNA polymerase and biotin-CTP (Affymetrix). Unincorporated nucleotides were removed using the RNeasy Mini kit (Qiagen) followed by an ethanol precipitation of the labeled target.
Target molecule: cRNA
Hyb protocol: Labeled cRNA targets were chemically fragmented at 95C for 35 min as per Affymetrix’s recommendations. The fragmented material was combined with Affymetrix hybridization controls: biotinylated control oligomer (B2) and biotinylated control cRNAs for BioB, BioC, BioD and CreX (Affymetrix) in hybridization buffer. Following the manufacturer’s recommendations, 6.5 µg of target were hybridized with the GeneChip Human Genome Focus array (Affymetrix) for 16 hours at 45C. Post-hybridization array processing was performed on the Fluidics Station 450 (Affymetrix); array staining and signal amplification were performed according to manufacturer’s recommendations.
|
06A-11H1H_Dpna_270JR
|
| Sample_geo_accession | GSM335423
| | Sample_status | Public on Jan 30 2009
| | Sample_submission_date | Oct 21 2008
| | Sample_last_update_date | Jan 30 2009
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Unfractionated whole blood collection and RNA isolation were performed using the PAXgene Blood RNA Isolation System (PreAnalytiX, a Qiagen BD Company, Valencia, CA, USA). With the approval of the OHSU Institutional Review Board, blood was collected from healthy donors using eight PAXGene tubes per individual (donor). Four tubes per donor were frozen for subsequent processing and four tubes per donor were processed fresh (see individual sample annotation for storage state details). Following RNA isolation, DNase-treatment was performed as per PAXGene manufacturer’s recommendation. Samples were then pooled within donor and within RNA storage state (fresh/frozen) resulting in two tubes per donor (fresh/frozen). RNA recovery and quality was then assessed by examining UV 260/280 absorbance ratios and RNA size distribution on RNA Nano LabChips (Agilent Technologies, Santa Clara, CA, USA) processed on the Agilent 2100 Bioanalyzer. For inclusion in this study, the RNA mass per donor had to be greater than 12 µg for both fresh and frozen blood aliquots. Samples from three of the four donors (Donors A, C, D) met the quantitative and qualitative criteria and were used in the microarray analysis.
| | Sample_growth_protocol_ch1 | n/a
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | [see characteristics field for details]
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Amplifications and labelings were performed for each donor using one of the following four methods with RNA extracted from fresh and frozen blood samples: Method 1 - Affymetrix one-cycle cDNA synthesis/Affymetrix in vitro-transcription (IVT) on whole blood RNA; Method 2 - Affymetrix one-cycle cDNA/Affymetrix IVT with globin peptide nucleic acids (PNAs) inclusion during cDNA synthesis; and Method 3- RNA pre-treated with Ambion GLOBINclear beads to reduce globin transcripts prior to labeling with the Affymetrix one-cycle cDNA synthesis/Affymetrix IVT. In Method 4- cDNA target was synthesized from whole blood RNA using the NuGEN Ovation Biotin RNA Amplification and Labeling System. See individual sample annotation for protocol details.
| | Sample_hyb_protocol | [see characteristics field for details]
| | Sample_scan_protocol | The distribution of fluorescent material on the processed array was determined using the Affymetrix 3000 GeneArray laser scanner with the 7G upgrade. Image inspection was performed manually immediately following each scan. The array image scan was processed with Affymetrix Microarray Suite, version 5.0, (MAS 5.0) software.
| | Sample_data_processing | Low-level analysis (background correction, normalization, and gene summarization) of microarray data was performed with both Affymetrix Microarray Suite 5.0 (MAS 5.0) and Robust Multi-Array Average (RMA). Individual arrays were analyzed and scaled with MAS 5.0 using manufacturer’s default thresholds for detection calls and with target scaling factor set as 275. For RMA analysis, arrays were normalized in four separate groups based on target labeling method using RMA implemented in Affymetrix Expression Console (version 1.1).
| | Sample_platform_id | GPL201
| | Sample_contact_name | Christina,A,Harrington
| | Sample_contact_email | harringc@ohsu.edu
| | Sample_contact_phone | 503-418-2737
| | Sample_contact_fax | 503-418-9381
| | Sample_contact_laboratory | Affymetrix Microarray Core Facility
| | Sample_contact_department | Gene Microarray Shared Resource
| | Sample_contact_institute | Oregon Health and Science University
| | Sample_contact_address | 3303 SW Bond Ave
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM335nnn/GSM335423/suppl/GSM335423.CEL.gz
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM335nnn/GSM335423/suppl/GSM335423.CHP.gz
| | Sample_series_id | GSE13292
| | Sample_data_row_count | 8793
| | |
|
GSM335424 | GPL201 |
|
A_frozen_Method 2
|
Whole blood, frozen, Globin PNAs_Affymetrix
|
Gender: M
Age: 31, Normal
Biosource: Peripheral whole blood
Donor ID: Donor A
Sample storage state: frozen
Sample protocol: Whole blood was incubated in a PAXGene tube (PreAnalytiX, a Qiagen BD Company, Valencia, CA, USA) for two hours and then transferred to a -80C freezer. RNA was isolated from the frozen PAXGene tube after storage at -80C for at least one week.
Treatment protocol: Affymetrix one-cycle cDNA synthesis/Affymetrix IVT with PNA treatment of total RNA (Globin PNAs_Affymetrix): The standard Affymetrix amplification and labeling was performed with the following modification: four peptide nucleic acid (PNA) oligonucleotides (Applied Biosystems, Foster City, CA, USA) designed to anneal to human α- and β-globin mRNA were added to the total RNA preparations immediately prior to initiation of the target synthesis step in order to bind globin mRNA and block cDNA synthesis. The PNA oligonucleotide stocks were prepared on day of use at concentrations recommended by Affymetrix (www.affymetrix.com/support/technical/byproduct.affx?product=bloodrna). Globin reduction PNA master mix was prepared by adding equal amounts of the globin PNA stocks. Five µg of total RNA were incubated with oligo-dT primer and 1 µl PNA master mix for 10 minutes at 70C prior to first strand cDNA synthesis.
Label protocol: Affymetrix One-Cycle: Target synthesis was performed following the Affymetrix GeneChip Expression Analysis Technical Manual, rev. 5 (http://www.affymetrix.com/support/technical/manual/expression_manual.affx). Using 1 µg of total RNA as input, messenger RNA was amplified and labeled in two steps. In the first step, mRNA was converted to double-stranded cDNA using Superscript Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) and an oligo-dT primer linked to a T7 RNA polymerase binding site sequence (Affymetrix, Santa Clara, CA, USA). Double-stranded cDNA was purified by phenol-chloroform-isoamyl alcohol extraction and a standard ethanol precipitation. In the second step, amplified and labeled cRNA (the target) was produced by a transcription reaction using T7 RNA polymerase and biotin-CTP (Affymetrix). Unincorporated nucleotides were removed using the RNeasy Mini kit (Qiagen) followed by an ethanol precipitation of the labeled target.
Target molecule: cRNA
Hyb protocol: Labeled cRNA targets were chemically fragmented at 95C for 35 min as per Affymetrix’s recommendations. The fragmented material was combined with Affymetrix hybridization controls: biotinylated control oligomer (B2) and biotinylated control cRNAs for BioB, BioC, BioD and CreX (Affymetrix) in hybridization buffer. Following the manufacturer’s recommendations, 6.5 µg of target were hybridized with the GeneChip Human Genome Focus array (Affymetrix) for 16 hours at 45C. Post-hybridization array processing was performed on the Fluidics Station 450 (Affymetrix); array staining and signal amplification were performed according to manufacturer’s recommendations.
|
08A-11H1H_Afpna_270JR
|
| Sample_geo_accession | GSM335424
| | Sample_status | Public on Jan 30 2009
| | Sample_submission_date | Oct 21 2008
| | Sample_last_update_date | Jan 30 2009
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Unfractionated whole blood collection and RNA isolation were performed using the PAXgene Blood RNA Isolation System (PreAnalytiX, a Qiagen BD Company, Valencia, CA, USA). With the approval of the OHSU Institutional Review Board, blood was collected from healthy donors using eight PAXGene tubes per individual (donor). Four tubes per donor were frozen for subsequent processing and four tubes per donor were processed fresh (see individual sample annotation for storage state details). Following RNA isolation, DNase-treatment was performed as per PAXGene manufacturer’s recommendation. Samples were then pooled within donor and within RNA storage state (fresh/frozen) resulting in two tubes per donor (fresh/frozen). RNA recovery and quality was then assessed by examining UV 260/280 absorbance ratios and RNA size distribution on RNA Nano LabChips (Agilent Technologies, Santa Clara, CA, USA) processed on the Agilent 2100 Bioanalyzer. For inclusion in this study, the RNA mass per donor had to be greater than 12 µg for both fresh and frozen blood aliquots. Samples from three of the four donors (Donors A, C, D) met the quantitative and qualitative criteria and were used in the microarray analysis.
| | Sample_growth_protocol_ch1 | n/a
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | [see characteristics field for details]
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Amplifications and labelings were performed for each donor using one of the following four methods with RNA extracted from fresh and frozen blood samples: Method 1 - Affymetrix one-cycle cDNA synthesis/Affymetrix in vitro-transcription (IVT) on whole blood RNA; Method 2 - Affymetrix one-cycle cDNA/Affymetrix IVT with globin peptide nucleic acids (PNAs) inclusion during cDNA synthesis; and Method 3- RNA pre-treated with Ambion GLOBINclear beads to reduce globin transcripts prior to labeling with the Affymetrix one-cycle cDNA synthesis/Affymetrix IVT. In Method 4- cDNA target was synthesized from whole blood RNA using the NuGEN Ovation Biotin RNA Amplification and Labeling System. See individual sample annotation for protocol details.
| | Sample_hyb_protocol | [see characteristics field for details]
| | Sample_scan_protocol | The distribution of fluorescent material on the processed array was determined using the Affymetrix 3000 GeneArray laser scanner with the 7G upgrade. Image inspection was performed manually immediately following each scan. The array image scan was processed with Affymetrix Microarray Suite, version 5.0, (MAS 5.0) software.
| | Sample_data_processing | Low-level analysis (background correction, normalization, and gene summarization) of microarray data was performed with both Affymetrix Microarray Suite 5.0 (MAS 5.0) and Robust Multi-Array Average (RMA). Individual arrays were analyzed and scaled with MAS 5.0 using manufacturer’s default thresholds for detection calls and with target scaling factor set as 275. For RMA analysis, arrays were normalized in four separate groups based on target labeling method using RMA implemented in Affymetrix Expression Console (version 1.1).
| | Sample_platform_id | GPL201
| | Sample_contact_name | Christina,A,Harrington
| | Sample_contact_email | harringc@ohsu.edu
| | Sample_contact_phone | 503-418-2737
| | Sample_contact_fax | 503-418-9381
| | Sample_contact_laboratory | Affymetrix Microarray Core Facility
| | Sample_contact_department | Gene Microarray Shared Resource
| | Sample_contact_institute | Oregon Health and Science University
| | Sample_contact_address | 3303 SW Bond Ave
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM335nnn/GSM335424/suppl/GSM335424.CEL.gz
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM335nnn/GSM335424/suppl/GSM335424.CHP.gz
| | Sample_series_id | GSE13292
| | Sample_data_row_count | 8793
| | |
|
GSM335425 | GPL201 |
|
C_frozen_Method 2
|
Whole blood, frozen, Globin PNAs_Affymetrix
|
Gender: M
Age: 25, Normal
Biosource: Peripheral whole blood
Donor ID: Donor C
Sample storage state: frozen
Sample protocol: Whole blood was incubated in a PAXGene tube (PreAnalytiX, a Qiagen BD Company, Valencia, CA, USA) for two hours and then transferred to a -80C freezer. RNA was isolated from the frozen PAXGene tube after storage at -80C for at least one week.
Treatment protocol: Affymetrix one-cycle cDNA synthesis/Affymetrix IVT with PNA treatment of total RNA (Globin PNAs_Affymetrix): The standard Affymetrix amplification and labeling was performed with the following modification: four peptide nucleic acid (PNA) oligonucleotides (Applied Biosystems, Foster City, CA, USA) designed to anneal to human α- and β-globin mRNA were added to the total RNA preparations immediately prior to initiation of the target synthesis step in order to bind globin mRNA and block cDNA synthesis. The PNA oligonucleotide stocks were prepared on day of use at concentrations recommended by Affymetrix (www.affymetrix.com/support/technical/byproduct.affx?product=bloodrna). Globin reduction PNA master mix was prepared by adding equal amounts of the globin PNA stocks. Five µg of total RNA were incubated with oligo-dT primer and 1 µl PNA master mix for 10 minutes at 70C prior to first strand cDNA synthesis.
Label protocol: Affymetrix One-Cycle: Target synthesis was performed following the Affymetrix GeneChip Expression Analysis Technical Manual, rev. 5 (http://www.affymetrix.com/support/technical/manual/expression_manual.affx). Using 1 µg of total RNA as input, messenger RNA was amplified and labeled in two steps. In the first step, mRNA was converted to double-stranded cDNA using Superscript Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) and an oligo-dT primer linked to a T7 RNA polymerase binding site sequence (Affymetrix, Santa Clara, CA, USA). Double-stranded cDNA was purified by phenol-chloroform-isoamyl alcohol extraction and a standard ethanol precipitation. In the second step, amplified and labeled cRNA (the target) was produced by a transcription reaction using T7 RNA polymerase and biotin-CTP (Affymetrix). Unincorporated nucleotides were removed using the RNeasy Mini kit (Qiagen) followed by an ethanol precipitation of the labeled target.
Target molecule: cRNA
Hyb protocol: Labeled cRNA targets were chemically fragmented at 95C for 35 min as per Affymetrix’s recommendations. The fragmented material was combined with Affymetrix hybridization controls: biotinylated control oligomer (B2) and biotinylated control cRNAs for BioB, BioC, BioD and CreX (Affymetrix) in hybridization buffer. Following the manufacturer’s recommendations, 6.5 µg of target were hybridized with the GeneChip Human Genome Focus array (Affymetrix) for 16 hours at 45C. Post-hybridization array processing was performed on the Fluidics Station 450 (Affymetrix); array staining and signal amplification were performed according to manufacturer’s recommendations.
|
10A-11H1H_Cfpna_270JR
|
| Sample_geo_accession | GSM335425
| | Sample_status | Public on Jan 30 2009
| | Sample_submission_date | Oct 21 2008
| | Sample_last_update_date | Jan 30 2009
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Unfractionated whole blood collection and RNA isolation were performed using the PAXgene Blood RNA Isolation System (PreAnalytiX, a Qiagen BD Company, Valencia, CA, USA). With the approval of the OHSU Institutional Review Board, blood was collected from healthy donors using eight PAXGene tubes per individual (donor). Four tubes per donor were frozen for subsequent processing and four tubes per donor were processed fresh (see individual sample annotation for storage state details). Following RNA isolation, DNase-treatment was performed as per PAXGene manufacturer’s recommendation. Samples were then pooled within donor and within RNA storage state (fresh/frozen) resulting in two tubes per donor (fresh/frozen). RNA recovery and quality was then assessed by examining UV 260/280 absorbance ratios and RNA size distribution on RNA Nano LabChips (Agilent Technologies, Santa Clara, CA, USA) processed on the Agilent 2100 Bioanalyzer. For inclusion in this study, the RNA mass per donor had to be greater than 12 µg for both fresh and frozen blood aliquots. Samples from three of the four donors (Donors A, C, D) met the quantitative and qualitative criteria and were used in the microarray analysis.
| | Sample_growth_protocol_ch1 | n/a
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | [see characteristics field for details]
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Amplifications and labelings were performed for each donor using one of the following four methods with RNA extracted from fresh and frozen blood samples: Method 1 - Affymetrix one-cycle cDNA synthesis/Affymetrix in vitro-transcription (IVT) on whole blood RNA; Method 2 - Affymetrix one-cycle cDNA/Affymetrix IVT with globin peptide nucleic acids (PNAs) inclusion during cDNA synthesis; and Method 3- RNA pre-treated with Ambion GLOBINclear beads to reduce globin transcripts prior to labeling with the Affymetrix one-cycle cDNA synthesis/Affymetrix IVT. In Method 4- cDNA target was synthesized from whole blood RNA using the NuGEN Ovation Biotin RNA Amplification and Labeling System. See individual sample annotation for protocol details.
| | Sample_hyb_protocol | [see characteristics field for details]
| | Sample_scan_protocol | The distribution of fluorescent material on the processed array was determined using the Affymetrix 3000 GeneArray laser scanner with the 7G upgrade. Image inspection was performed manually immediately following each scan. The array image scan was processed with Affymetrix Microarray Suite, version 5.0, (MAS 5.0) software.
| | Sample_data_processing | Low-level analysis (background correction, normalization, and gene summarization) of microarray data was performed with both Affymetrix Microarray Suite 5.0 (MAS 5.0) and Robust Multi-Array Average (RMA). Individual arrays were analyzed and scaled with MAS 5.0 using manufacturer’s default thresholds for detection calls and with target scaling factor set as 275. For RMA analysis, arrays were normalized in four separate groups based on target labeling method using RMA implemented in Affymetrix Expression Console (version 1.1).
| | Sample_platform_id | GPL201
| | Sample_contact_name | Christina,A,Harrington
| | Sample_contact_email | harringc@ohsu.edu
| | Sample_contact_phone | 503-418-2737
| | Sample_contact_fax | 503-418-9381
| | Sample_contact_laboratory | Affymetrix Microarray Core Facility
| | Sample_contact_department | Gene Microarray Shared Resource
| | Sample_contact_institute | Oregon Health and Science University
| | Sample_contact_address | 3303 SW Bond Ave
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM335nnn/GSM335425/suppl/GSM335425.CEL.gz
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM335nnn/GSM335425/suppl/GSM335425.CHP.gz
| | Sample_series_id | GSE13292
| | Sample_data_row_count | 8793
| | |
|
GSM335426 | GPL201 |
|
D_frozen_Method 2
|
Whole blood, frozen, Globin PNAs_Affymetrix
|
Gender: M
Age: 24, Normal
Biosource: Peripheral whole blood
Donor ID: Donor D
Sample storage state: frozen
Sample protocol: Whole blood was incubated in a PAXGene tube (PreAnalytiX, a Qiagen BD Company, Valencia, CA, USA) for two hours and then transferred to a -80C freezer. RNA was isolated from the frozen PAXGene tube after storage at -80C for at least one week.
Treatment protocol: Affymetrix one-cycle cDNA synthesis/Affymetrix IVT with PNA treatment of total RNA (Globin PNAs_Affymetrix): The standard Affymetrix amplification and labeling was performed with the following modification: four peptide nucleic acid (PNA) oligonucleotides (Applied Biosystems, Foster City, CA, USA) designed to anneal to human α- and β-globin mRNA were added to the total RNA preparations immediately prior to initiation of the target synthesis step in order to bind globin mRNA and block cDNA synthesis. The PNA oligonucleotide stocks were prepared on day of use at concentrations recommended by Affymetrix (www.affymetrix.com/support/technical/byproduct.affx?product=bloodrna). Globin reduction PNA master mix was prepared by adding equal amounts of the globin PNA stocks. Five µg of total RNA were incubated with oligo-dT primer and 1 µl PNA master mix for 10 minutes at 70C prior to first strand cDNA synthesis.
Label protocol: Affymetrix One-Cycle: Target synthesis was performed following the Affymetrix GeneChip Expression Analysis Technical Manual, rev. 5 (http://www.affymetrix.com/support/technical/manual/expression_manual.affx). Using 1 µg of total RNA as input, messenger RNA was amplified and labeled in two steps. In the first step, mRNA was converted to double-stranded cDNA using Superscript Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) and an oligo-dT primer linked to a T7 RNA polymerase binding site sequence (Affymetrix, Santa Clara, CA, USA). Double-stranded cDNA was purified by phenol-chloroform-isoamyl alcohol extraction and a standard ethanol precipitation. In the second step, amplified and labeled cRNA (the target) was produced by a transcription reaction using T7 RNA polymerase and biotin-CTP (Affymetrix). Unincorporated nucleotides were removed using the RNeasy Mini kit (Qiagen) followed by an ethanol precipitation of the labeled target.
Target molecule: cRNA
Hyb protocol: Labeled cRNA targets were chemically fragmented at 95C for 35 min as per Affymetrix’s recommendations. The fragmented material was combined with Affymetrix hybridization controls: biotinylated control oligomer (B2) and biotinylated control cRNAs for BioB, BioC, BioD and CreX (Affymetrix) in hybridization buffer. Following the manufacturer’s recommendations, 6.5 µg of target were hybridized with the GeneChip Human Genome Focus array (Affymetrix) for 16 hours at 45C. Post-hybridization array processing was performed on the Fluidics Station 450 (Affymetrix); array staining and signal amplification were performed according to manufacturer’s recommendations.
|
12A-11H1H_Dfpna_270JR
|
| Sample_geo_accession | GSM335426
| | Sample_status | Public on Jan 30 2009
| | Sample_submission_date | Oct 21 2008
| | Sample_last_update_date | Jan 30 2009
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Unfractionated whole blood collection and RNA isolation were performed using the PAXgene Blood RNA Isolation System (PreAnalytiX, a Qiagen BD Company, Valencia, CA, USA). With the approval of the OHSU Institutional Review Board, blood was collected from healthy donors using eight PAXGene tubes per individual (donor). Four tubes per donor were frozen for subsequent processing and four tubes per donor were processed fresh (see individual sample annotation for storage state details). Following RNA isolation, DNase-treatment was performed as per PAXGene manufacturer’s recommendation. Samples were then pooled within donor and within RNA storage state (fresh/frozen) resulting in two tubes per donor (fresh/frozen). RNA recovery and quality was then assessed by examining UV 260/280 absorbance ratios and RNA size distribution on RNA Nano LabChips (Agilent Technologies, Santa Clara, CA, USA) processed on the Agilent 2100 Bioanalyzer. For inclusion in this study, the RNA mass per donor had to be greater than 12 µg for both fresh and frozen blood aliquots. Samples from three of the four donors (Donors A, C, D) met the quantitative and qualitative criteria and were used in the microarray analysis.
| | Sample_growth_protocol_ch1 | n/a
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | [see characteristics field for details]
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Amplifications and labelings were performed for each donor using one of the following four methods with RNA extracted from fresh and frozen blood samples: Method 1 - Affymetrix one-cycle cDNA synthesis/Affymetrix in vitro-transcription (IVT) on whole blood RNA; Method 2 - Affymetrix one-cycle cDNA/Affymetrix IVT with globin peptide nucleic acids (PNAs) inclusion during cDNA synthesis; and Method 3- RNA pre-treated with Ambion GLOBINclear beads to reduce globin transcripts prior to labeling with the Affymetrix one-cycle cDNA synthesis/Affymetrix IVT. In Method 4- cDNA target was synthesized from whole blood RNA using the NuGEN Ovation Biotin RNA Amplification and Labeling System. See individual sample annotation for protocol details.
| | Sample_hyb_protocol | [see characteristics field for details]
| | Sample_scan_protocol | The distribution of fluorescent material on the processed array was determined using the Affymetrix 3000 GeneArray laser scanner with the 7G upgrade. Image inspection was performed manually immediately following each scan. The array image scan was processed with Affymetrix Microarray Suite, version 5.0, (MAS 5.0) software.
| | Sample_data_processing | Low-level analysis (background correction, normalization, and gene summarization) of microarray data was performed with both Affymetrix Microarray Suite 5.0 (MAS 5.0) and Robust Multi-Array Average (RMA). Individual arrays were analyzed and scaled with MAS 5.0 using manufacturer’s default thresholds for detection calls and with target scaling factor set as 275. For RMA analysis, arrays were normalized in four separate groups based on target labeling method using RMA implemented in Affymetrix Expression Console (version 1.1).
| | Sample_platform_id | GPL201
| | Sample_contact_name | Christina,A,Harrington
| | Sample_contact_email | harringc@ohsu.edu
| | Sample_contact_phone | 503-418-2737
| | Sample_contact_fax | 503-418-9381
| | Sample_contact_laboratory | Affymetrix Microarray Core Facility
| | Sample_contact_department | Gene Microarray Shared Resource
| | Sample_contact_institute | Oregon Health and Science University
| | Sample_contact_address | 3303 SW Bond Ave
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM335nnn/GSM335426/suppl/GSM335426.CEL.gz
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM335nnn/GSM335426/suppl/GSM335426.CHP.gz
| | Sample_series_id | GSE13292
| | Sample_data_row_count | 8793
| | |
|
GSM335427 | GPL201 |
|
A_fresh_Method 3
|
Whole blood, fresh, GLOBINclear_Affymetrix
|
Gender: M
Age: 31, Normal
Biosource: Peripheral whole blood
Donor ID: Donor A
Sample storage state: fresh
Sample protocol: Whole blood was incubated in a PAXGene tube (PreAnalytiX, a Qiagen BD Company, Valencia, CA, USA) for two hours and then RNA was isolated from the fresh sample.
Treatment protocol: Affymetrix one-cycle cDNA synthesis/Affymetrix IVT with GLOBINclear treated total RNA (GLOBINclear_Affymetrix): Globin mRNA depletion was performed in three steps using the GLOBINclear Kit (Ambion, Austin, TX, USA). First, species-specific biotinylated oligonucleotides complementary to human α- and β- globin mRNA were hybridized with total RNA (5 µg). Second, magnetic streptavidin-coated beads were added to bind the biotinylated-oligonucleotide:globin RNA complexes. Finally, a magnet was used to remove beads with the selectively bound α- and β-globin transcripts. After globin mRNA depletion, RNA quantity was determined by UV absorbance and RNA integrity was assessed using RNA Nano LabChips as described above. RNA recovery following GLOBINclear treatment ranged from 2-5 µg.
Label protocol: Affymetrix One-Cycle: Target synthesis was performed following the Affymetrix GeneChip Expression Analysis Technical Manual, rev. 5 (http://www.affymetrix.com/support/technical/manual/expression_manual.affx). Using 1 µg of total RNA as input, messenger RNA was amplified and labeled in two steps. In the first step, mRNA was converted to double-stranded cDNA using Superscript Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) and an oligo-dT primer linked to a T7 RNA polymerase binding site sequence (Affymetrix, Santa Clara, CA, USA). Double-stranded cDNA was purified by phenol-chloroform-isoamyl alcohol extraction and a standard ethanol precipitation. In the second step, amplified and labeled cRNA (the target) was produced by a transcription reaction using T7 RNA polymerase and biotin-CTP (Affymetrix). Unincorporated nucleotides were removed using the RNeasy Mini kit (Qiagen) followed by an ethanol precipitation of the labeled target.
Target molecule: cRNA
Hyb protocol: Labeled cRNA targets were chemically fragmented at 95C for 35 min as per Affymetrix’s recommendations. The fragmented material was combined with Affymetrix hybridization controls: biotinylated control oligomer (B2) and biotinylated control cRNAs for BioB, BioC, BioD and CreX (Affymetrix) in hybridization buffer. Following the manufacturer’s recommendations, 6.5 µg of target were hybridized with the GeneChip Human Genome Focus array (Affymetrix) for 16 hours at 45C. Post-hybridization array processing was performed on the Fluidics Station 450 (Affymetrix); array staining and signal amplification were performed according to manufacturer’s recommendations.
|
13A-11H1H_Adep_270JR
|
| Sample_geo_accession | GSM335427
| | Sample_status | Public on Jan 30 2009
| | Sample_submission_date | Oct 21 2008
| | Sample_last_update_date | Jan 30 2009
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Unfractionated whole blood collection and RNA isolation were performed using the PAXgene Blood RNA Isolation System (PreAnalytiX, a Qiagen BD Company, Valencia, CA, USA). With the approval of the OHSU Institutional Review Board, blood was collected from healthy donors using eight PAXGene tubes per individual (donor). Four tubes per donor were frozen for subsequent processing and four tubes per donor were processed fresh (see individual sample annotation for storage state details). Following RNA isolation, DNase-treatment was performed as per PAXGene manufacturer’s recommendation. Samples were then pooled within donor and within RNA storage state (fresh/frozen) resulting in two tubes per donor (fresh/frozen). RNA recovery and quality was then assessed by examining UV 260/280 absorbance ratios and RNA size distribution on RNA Nano LabChips (Agilent Technologies, Santa Clara, CA, USA) processed on the Agilent 2100 Bioanalyzer. For inclusion in this study, the RNA mass per donor had to be greater than 12 µg for both fresh and frozen blood aliquots. Samples from three of the four donors (Donors A, C, D) met the quantitative and qualitative criteria and were used in the microarray analysis.
| | Sample_growth_protocol_ch1 | n/a
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | [see characteristics field for details]
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Amplifications and labelings were performed for each donor using one of the following four methods with RNA extracted from fresh and frozen blood samples: Method 1 - Affymetrix one-cycle cDNA synthesis/Affymetrix in vitro-transcription (IVT) on whole blood RNA; Method 2 - Affymetrix one-cycle cDNA/Affymetrix IVT with globin peptide nucleic acids (PNAs) inclusion during cDNA synthesis; and Method 3- RNA pre-treated with Ambion GLOBINclear beads to reduce globin transcripts prior to labeling with the Affymetrix one-cycle cDNA synthesis/Affymetrix IVT. In Method 4- cDNA target was synthesized from whole blood RNA using the NuGEN Ovation Biotin RNA Amplification and Labeling System. See individual sample annotation for protocol details.
| | Sample_hyb_protocol | [see characteristics field for details]
| | Sample_scan_protocol | The distribution of fluorescent material on the processed array was determined using the Affymetrix 3000 GeneArray laser scanner with the 7G upgrade. Image inspection was performed manually immediately following each scan. The array image scan was processed with Affymetrix Microarray Suite, version 5.0, (MAS 5.0) software.
| | Sample_data_processing | Low-level analysis (background correction, normalization, and gene summarization) of microarray data was performed with both Affymetrix Microarray Suite 5.0 (MAS 5.0) and Robust Multi-Array Average (RMA). Individual arrays were analyzed and scaled with MAS 5.0 using manufacturer’s default thresholds for detection calls and with target scaling factor set as 275. For RMA analysis, arrays were normalized in four separate groups based on target labeling method using RMA implemented in Affymetrix Expression Console (version 1.1).
| | Sample_platform_id | GPL201
| | Sample_contact_name | Christina,A,Harrington
| | Sample_contact_email | harringc@ohsu.edu
| | Sample_contact_phone | 503-418-2737
| | Sample_contact_fax | 503-418-9381
| | Sample_contact_laboratory | Affymetrix Microarray Core Facility
| | Sample_contact_department | Gene Microarray Shared Resource
| | Sample_contact_institute | Oregon Health and Science University
| | Sample_contact_address | 3303 SW Bond Ave
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM335nnn/GSM335427/suppl/GSM335427.CEL.gz
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM335nnn/GSM335427/suppl/GSM335427.CHP.gz
| | Sample_series_id | GSE13292
| | Sample_data_row_count | 8793
| | |
|
GSM335428 | GPL201 |
|
C_fresh_Method 3
|
Whole blood, fresh, GLOBINclear_Affymetrix
|
Gender: M
Age: 25, Normal
Biosource: Peripheral whole blood
Donor ID: Donor C
Sample storage state: fresh
Sample protocol: Whole blood was incubated in a PAXGene tube (PreAnalytiX, a Qiagen BD Company, Valencia, CA, USA) for two hours and then RNA was isolated from the fresh sample.
Treatment protocol: Affymetrix one-cycle cDNA synthesis/Affymetrix IVT with GLOBINclear treated total RNA (GLOBINclear_Affymetrix): Globin mRNA depletion was performed in three steps using the GLOBINclear Kit (Ambion, Austin, TX, USA). First, species-specific biotinylated oligonucleotides complementary to human α- and β- globin mRNA were hybridized with total RNA (5 µg). Second, magnetic streptavidin-coated beads were added to bind the biotinylated-oligonucleotide:globin RNA complexes. Finally, a magnet was used to remove beads with the selectively bound α- and β-globin transcripts. After globin mRNA depletion, RNA quantity was determined by UV absorbance and RNA integrity was assessed using RNA Nano LabChips as described above. RNA recovery following GLOBINclear treatment ranged from 2-5 µg. Label protocol: Affymetrix One-Cycle: Target synthesis was performed following the Affymetrix GeneChip Expression Analysis Technical Manual, rev. 5 (http://www.affymetrix.com/support/technical/manual/expression_manual.affx). Using 1 µg of total RNA as input, messenger RNA was amplified and labeled in two steps. In the first step, mRNA was converted to double-stranded cDNA using Superscript Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) and an oligo-dT primer linked to a T7 RNA polymerase binding site sequence (Affymetrix, Santa Clara, CA, USA). Double-stranded cDNA was purified by phenol-chloroform-isoamyl alcohol extraction and a standard ethanol precipitation. In the second step, amplified and labeled cRNA (the target) was produced by a transcription reaction using T7 RNA polymerase and biotin-CTP (Affymetrix). Unincorporated nucleotides were removed using the RNeasy Mini kit (Qiagen) followed by an ethanol precipitation of the labeled target.
Target molecule: cRNA
Hyb protocol: Labeled cRNA targets were chemically fragmented at 95C for 35 min as per Affymetrix’s recommendations. The fragmented material was combined with Affymetrix hybridization controls: biotinylated control oligomer (B2) and biotinylated control cRNAs for BioB, BioC, BioD and CreX (Affymetrix) in hybridization buffer. Following the manufacturer’s recommendations, 6.5 µg of target were hybridized with the GeneChip Human Genome Focus array (Affymetrix) for 16 hours at 45C. Post-hybridization array processing was performed on the Fluidics Station 450 (Affymetrix); array staining and signal amplification were performed according to manufacturer’s recommendations.
|
14A-11H1H_Cdep_270JR
|
| Sample_geo_accession | GSM335428
| | Sample_status | Public on Jan 30 2009
| | Sample_submission_date | Oct 21 2008
| | Sample_last_update_date | Jan 30 2009
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Unfractionated whole blood collection and RNA isolation were performed using the PAXgene Blood RNA Isolation System (PreAnalytiX, a Qiagen BD Company, Valencia, CA, USA). With the approval of the OHSU Institutional Review Board, blood was collected from healthy donors using eight PAXGene tubes per individual (donor). Four tubes per donor were frozen for subsequent processing and four tubes per donor were processed fresh (see individual sample annotation for storage state details). Following RNA isolation, DNase-treatment was performed as per PAXGene manufacturer’s recommendation. Samples were then pooled within donor and within RNA storage state (fresh/frozen) resulting in two tubes per donor (fresh/frozen). RNA recovery and quality was then assessed by examining UV 260/280 absorbance ratios and RNA size distribution on RNA Nano LabChips (Agilent Technologies, Santa Clara, CA, USA) processed on the Agilent 2100 Bioanalyzer. For inclusion in this study, the RNA mass per donor had to be greater than 12 µg for both fresh and frozen blood aliquots. Samples from three of the four donors (Donors A, C, D) met the quantitative and qualitative criteria and were used in the microarray analysis.
| | Sample_growth_protocol_ch1 | n/a
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | [see characteristics field for details]
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Amplifications and labelings were performed for each donor using one of the following four methods with RNA extracted from fresh and frozen blood samples: Method 1 - Affymetrix one-cycle cDNA synthesis/Affymetrix in vitro-transcription (IVT) on whole blood RNA; Method 2 - Affymetrix one-cycle cDNA/Affymetrix IVT with globin peptide nucleic acids (PNAs) inclusion during cDNA synthesis; and Method 3- RNA pre-treated with Ambion GLOBINclear beads to reduce globin transcripts prior to labeling with the Affymetrix one-cycle cDNA synthesis/Affymetrix IVT. In Method 4- cDNA target was synthesized from whole blood RNA using the NuGEN Ovation Biotin RNA Amplification and Labeling System. See individual sample annotation for protocol details.
| | Sample_hyb_protocol | [see characteristics field for details]
| | Sample_scan_protocol | The distribution of fluorescent material on the processed array was determined using the Affymetrix 3000 GeneArray laser scanner with the 7G upgrade. Image inspection was performed manually immediately following each scan. The array image scan was processed with Affymetrix Microarray Suite, version 5.0, (MAS 5.0) software.
| | Sample_data_processing | Low-level analysis (background correction, normalization, and gene summarization) of microarray data was performed with both Affymetrix Microarray Suite 5.0 (MAS 5.0) and Robust Multi-Array Average (RMA). Individual arrays were analyzed and scaled with MAS 5.0 using manufacturer’s default thresholds for detection calls and with target scaling factor set as 275. For RMA analysis, arrays were normalized in four separate groups based on target labeling method using RMA implemented in Affymetrix Expression Console (version 1.1).
| | Sample_platform_id | GPL201
| | Sample_contact_name | Christina,A,Harrington
| | Sample_contact_email | harringc@ohsu.edu
| | Sample_contact_phone | 503-418-2737
| | Sample_contact_fax | 503-418-9381
| | Sample_contact_laboratory | Affymetrix Microarray Core Facility
| | Sample_contact_department | Gene Microarray Shared Resource
| | Sample_contact_institute | Oregon Health and Science University
| | Sample_contact_address | 3303 SW Bond Ave
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM335nnn/GSM335428/suppl/GSM335428.CEL.gz
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM335nnn/GSM335428/suppl/GSM335428.CHP.gz
| | Sample_series_id | GSE13292
| | Sample_data_row_count | 8793
| | |
|
GSM335429 | GPL201 |
|
D_fresh_Method 3
|
Whole blood, fresh, GLOBINclear_Affymetrix
|
Gender: M
Age: 24, Normal
Biosource: Peripheral whole blood
Donor ID: Donor D
Sample storage state: fresh
Sample protocol: Whole blood was incubated in a PAXGene tube (PreAnalytiX, a Qiagen BD Company, Valencia, CA, USA) for two hours and then RNA was isolated from the fresh sample.
Treatment protocol: Affymetrix one-cycle cDNA synthesis/Affymetrix IVT with GLOBINclear treated total RNA (GLOBINclear_Affymetrix): Globin mRNA depletion was performed in three steps using the GLOBINclear Kit (Ambion, Austin, TX, USA). First, species-specific biotinylated oligonucleotides complementary to human α- and β- globin mRNA were hybridized with total RNA (5 µg). Second, magnetic streptavidin-coated beads were added to bind the biotinylated-oligonucleotide:globin RNA complexes. Finally, a magnet was used to remove beads with the selectively bound α- and β-globin transcripts. After globin mRNA depletion, RNA quantity was determined by UV absorbance and RNA integrity was assessed using RNA Nano LabChips as described above. RNA recovery following GLOBINclear treatment ranged from 2-5 µg.
Label protocol: Affymetrix One-Cycle: Target synthesis was performed following the Affymetrix GeneChip Expression Analysis Technical Manual, rev. 5 (http://www.affymetrix.com/support/technical/manual/expression_manual.affx). Using 1 µg of total RNA as input, messenger RNA was amplified and labeled in two steps. In the first step, mRNA was converted to double-stranded cDNA using Superscript Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) and an oligo-dT primer linked to a T7 RNA polymerase binding site sequence (Affymetrix, Santa Clara, CA, USA). Double-stranded cDNA was purified by phenol-chloroform-isoamyl alcohol extraction and a standard ethanol precipitation. In the second step, amplified and labeled cRNA (the target) was produced by a transcription reaction using T7 RNA polymerase and biotin-CTP (Affymetrix). Unincorporated nucleotides were removed using the RNeasy Mini kit (Qiagen) followed by an ethanol precipitation of the labeled target.
Target molecule: cRNA
Hyb protocol: Labeled cRNA targets were chemically fragmented at 95C for 35 min as per Affymetrix’s recommendations. The fragmented material was combined with Affymetrix hybridization controls: biotinylated control oligomer (B2) and biotinylated control cRNAs for BioB, BioC, BioD and CreX (Affymetrix) in hybridization buffer. Following the manufacturer’s recommendations, 6.5 µg of target were hybridized with the GeneChip Human Genome Focus array (Affymetrix) for 16 hours at 45C. Post-hybridization array processing was performed on the Fluidics Station 450 (Affymetrix); array staining and signal amplification were performed according to manufacturer’s recommendations.
|
15A-11H1H_Ddep_270JR
|
| Sample_geo_accession | GSM335429
| | Sample_status | Public on Jan 30 2009
| | Sample_submission_date | Oct 21 2008
| | Sample_last_update_date | Jan 30 2009
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Unfractionated whole blood collection and RNA isolation were performed using the PAXgene Blood RNA Isolation System (PreAnalytiX, a Qiagen BD Company, Valencia, CA, USA). With the approval of the OHSU Institutional Review Board, blood was collected from healthy donors using eight PAXGene tubes per individual (donor). Four tubes per donor were frozen for subsequent processing and four tubes per donor were processed fresh (see individual sample annotation for storage state details). Following RNA isolation, DNase-treatment was performed as per PAXGene manufacturer’s recommendation. Samples were then pooled within donor and within RNA storage state (fresh/frozen) resulting in two tubes per donor (fresh/frozen). RNA recovery and quality was then assessed by examining UV 260/280 absorbance ratios and RNA size distribution on RNA Nano LabChips (Agilent Technologies, Santa Clara, CA, USA) processed on the Agilent 2100 Bioanalyzer. For inclusion in this study, the RNA mass per donor had to be greater than 12 µg for both fresh and frozen blood aliquots. Samples from three of the four donors (Donors A, C, D) met the quantitative and qualitative criteria and were used in the microarray analysis.
| | Sample_growth_protocol_ch1 | n/a
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | [see characteristics field for details]
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Amplifications and labelings were performed for each donor using one of the following four methods with RNA extracted from fresh and frozen blood samples: Method 1 - Affymetrix one-cycle cDNA synthesis/Affymetrix in vitro-transcription (IVT) on whole blood RNA; Method 2 - Affymetrix one-cycle cDNA/Affymetrix IVT with globin peptide nucleic acids (PNAs) inclusion during cDNA synthesis; and Method 3- RNA pre-treated with Ambion GLOBINclear beads to reduce globin transcripts prior to labeling with the Affymetrix one-cycle cDNA synthesis/Affymetrix IVT. In Method 4- cDNA target was synthesized from whole blood RNA using the NuGEN Ovation Biotin RNA Amplification and Labeling System. See individual sample annotation for protocol details.
| | Sample_hyb_protocol | [see characteristics field for details]
| | Sample_scan_protocol | The distribution of fluorescent material on the processed array was determined using the Affymetrix 3000 GeneArray laser scanner with the 7G upgrade. Image inspection was performed manually immediately following each scan. The array image scan was processed with Affymetrix Microarray Suite, version 5.0, (MAS 5.0) software.
| | Sample_data_processing | Low-level analysis (background correction, normalization, and gene summarization) of microarray data was performed with both Affymetrix Microarray Suite 5.0 (MAS 5.0) and Robust Multi-Array Average (RMA). Individual arrays were analyzed and scaled with MAS 5.0 using manufacturer’s default thresholds for detection calls and with target scaling factor set as 275. For RMA analysis, arrays were normalized in four separate groups based on target labeling method using RMA implemented in Affymetrix Expression Console (version 1.1).
| | Sample_platform_id | GPL201
| | Sample_contact_name | Christina,A,Harrington
| | Sample_contact_email | harringc@ohsu.edu
| | Sample_contact_phone | 503-418-2737
| | Sample_contact_fax | 503-418-9381
| | Sample_contact_laboratory | Affymetrix Microarray Core Facility
| | Sample_contact_department | Gene Microarray Shared Resource
| | Sample_contact_institute | Oregon Health and Science University
| | Sample_contact_address | 3303 SW Bond Ave
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM335nnn/GSM335429/suppl/GSM335429.CEL.gz
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM335nnn/GSM335429/suppl/GSM335429.CHP.gz
| | Sample_series_id | GSE13292
| | Sample_data_row_count | 8793
| | |
|
GSM335430 | GPL201 |
|
A_frozen_Method 3
|
Whole blood, frozen, GLOBINclear_Affymetrix
|
Gender: M
Age: 31, Normal
Biosource: Peripheral whole blood
Donor ID: Donor A
Sample storage state: frozen
Sample protocol: Whole blood was incubated in a PAXGene tube (PreAnalytiX, a Qiagen BD Company, Valencia, CA, USA) for two hours and then transferred to a -80C freezer. RNA was isolated from the frozen PAXGene tube after storage at -80C for at least one week. Treatment protocol: Affymetrix one-cycle cDNA synthesis/Affymetrix IVT with GLOBINclear treated total RNA (GLOBINclear_Affymetrix): Globin mRNA depletion was performed in three steps using the GLOBINclear Kit (Ambion, Austin, TX, USA). First, species-specific biotinylated oligonucleotides complementary to human α- and β-globin mRNA were hybridized with total RNA (5 µg). Second, magnetic streptavidin-coated beads were added to bind the biotinylated-oligonucleotide:globin RNA complexes. Finally, a magnet was used to remove beads with the selectively bound α- and β-globin transcripts. After globin mRNA depletion, RNA quantity was determined by UV absorbance and RNA integrity was assessed using RNA Nano LabChips as described above. RNA recovery following GLOBINclear treatment ranged from 2-5 µg. Label protocol: Affymetrix One-Cycle: Target synthesis was performed following the Affymetrix GeneChip Expression Analysis Technical Manual, rev. 5 (http://www.affymetrix.com/support/technical/manual/expression_manual.affx). Using 1 µg of total RNA as input, messenger RNA was amplified and labeled in two steps. In the first step, mRNA was converted to double-stranded cDNA using Superscript Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) and an oligo-dT primer linked to a T7 RNA polymerase binding site sequence (Affymetrix, Santa Clara, CA, USA). Double-stranded cDNA was purified by phenol-chloroform-isoamyl alcohol extraction and a standard ethanol precipitation. In the second step, amplified and labeled cRNA (the target) was produced by a transcription reaction using T7 RNA polymerase and biotin-CTP (Affymetrix). Unincorporated nucleotides were removed using the RNeasy Mini kit (Qiagen) followed by an ethanol precipitation of the labeled target.
Target molecule: cRNA
Hyb protocol: Labeled cRNA targets were chemically fragmented at 95C for 35 min as per Affymetrix’s recommendations. The fragmented material was combined with Affymetrix hybridization controls: biotinylated control oligomer (B2) and biotinylated control cRNAs for BioB, BioC, BioD and CreX (Affymetrix) in hybridization buffer. Following the manufacturer’s recommendations, 6.5 µg of target were hybridized with the GeneChip Human Genome Focus array (Affymetrix) for 16 hours at 45C. Post-hybridization array processing was performed on the Fluidics Station 450 (Affymetrix); array staining and signal amplification were performed according to manufacturer’s recommendations.
|
16A-11H1H_Afdep_270JR
|
| Sample_geo_accession | GSM335430
| | Sample_status | Public on Jan 30 2009
| | Sample_submission_date | Oct 21 2008
| | Sample_last_update_date | Jan 30 2009
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Unfractionated whole blood collection and RNA isolation were performed using the PAXgene Blood RNA Isolation System (PreAnalytiX, a Qiagen BD Company, Valencia, CA, USA). With the approval of the OHSU Institutional Review Board, blood was collected from healthy donors using eight PAXGene tubes per individual (donor). Four tubes per donor were frozen for subsequent processing and four tubes per donor were processed fresh (see individual sample annotation for storage state details). Following RNA isolation, DNase-treatment was performed as per PAXGene manufacturer’s recommendation. Samples were then pooled within donor and within RNA storage state (fresh/frozen) resulting in two tubes per donor (fresh/frozen). RNA recovery and quality was then assessed by examining UV 260/280 absorbance ratios and RNA size distribution on RNA Nano LabChips (Agilent Technologies, Santa Clara, CA, USA) processed on the Agilent 2100 Bioanalyzer. For inclusion in this study, the RNA mass per donor had to be greater than 12 µg for both fresh and frozen blood aliquots. Samples from three of the four donors (Donors A, C, D) met the quantitative and qualitative criteria and were used in the microarray analysis.
| | Sample_growth_protocol_ch1 | n/a
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | [see characteristics field for details]
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Amplifications and labelings were performed for each donor using one of the following four methods with RNA extracted from fresh and frozen blood samples: Method 1 - Affymetrix one-cycle cDNA synthesis/Affymetrix in vitro-transcription (IVT) on whole blood RNA; Method 2 - Affymetrix one-cycle cDNA/Affymetrix IVT with globin peptide nucleic acids (PNAs) inclusion during cDNA synthesis; and Method 3- RNA pre-treated with Ambion GLOBINclear beads to reduce globin transcripts prior to labeling with the Affymetrix one-cycle cDNA synthesis/Affymetrix IVT. In Method 4- cDNA target was synthesized from whole blood RNA using the NuGEN Ovation Biotin RNA Amplification and Labeling System. See individual sample annotation for protocol details.
| | Sample_hyb_protocol | [see characteristics field for details]
| | Sample_scan_protocol | The distribution of fluorescent material on the processed array was determined using the Affymetrix 3000 GeneArray laser scanner with the 7G upgrade. Image inspection was performed manually immediately following each scan. The array image scan was processed with Affymetrix Microarray Suite, version 5.0, (MAS 5.0) software.
| | Sample_data_processing | Low-level analysis (background correction, normalization, and gene summarization) of microarray data was performed with both Affymetrix Microarray Suite 5.0 (MAS 5.0) and Robust Multi-Array Average (RMA). Individual arrays were analyzed and scaled with MAS 5.0 using manufacturer’s default thresholds for detection calls and with target scaling factor set as 275. For RMA analysis, arrays were normalized in four separate groups based on target labeling method using RMA implemented in Affymetrix Expression Console (version 1.1).
| | Sample_platform_id | GPL201
| | Sample_contact_name | Christina,A,Harrington
| | Sample_contact_email | harringc@ohsu.edu
| | Sample_contact_phone | 503-418-2737
| | Sample_contact_fax | 503-418-9381
| | Sample_contact_laboratory | Affymetrix Microarray Core Facility
| | Sample_contact_department | Gene Microarray Shared Resource
| | Sample_contact_institute | Oregon Health and Science University
| | Sample_contact_address | 3303 SW Bond Ave
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM335nnn/GSM335430/suppl/GSM335430.CEL.gz
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM335nnn/GSM335430/suppl/GSM335430.CHP.gz
| | Sample_series_id | GSE13292
| | Sample_data_row_count | 8793
| | |
|
GSM335431 | GPL201 |
|
C_frozen_Method 3
|
Peripheral whole blood, frozen, GLOBINclear_Affymetrix
|
Gender: M
Age: 25, Normal
Biosource: Peripheral whole blood
Donor ID: Donor C
Sample storage state: frozen
Sample protocol: Whole blood was incubated in a PAXGene tube (PreAnalytiX, a Qiagen BD Company, Valencia, CA, USA) for two hours and then transferred to a -80C freezer. RNA was isolated from the frozen PAXGene tube after storage at -80C for at least one week. Treatment protocol: Affymetrix one-cycle cDNA synthesis/Affymetrix IVT with GLOBINclear treated total RNA (GLOBINclear_Affymetrix): Globin mRNA depletion was performed in three steps using the GLOBINclear Kit (Ambion, Austin, TX, USA). First, species-specific biotinylated oligonucleotides complementary to human α- and β-globin mRNA were hybridized with total RNA (5 µg). Second, magnetic streptavidin-coated beads were added to bind the biotinylated-oligonucleotide:globin RNA complexes. Finally, a magnet was used to remove beads with the selectively bound α- and β-globin transcripts. After globin mRNA depletion, RNA quantity was determined by UV absorbance and RNA integrity was assessed using RNA Nano LabChips as described above. RNA recovery following GLOBINclear treatment ranged from 2-5 µg. Label protocol: Affymetrix One-Cycle: Target synthesis was performed following the Affymetrix GeneChip Expression Analysis Technical Manual, rev. 5 (http://www.affymetrix.com/support/technical/manual/expression_manual.affx). Using 1 µg of total RNA as input, messenger RNA was amplified and labeled in two steps. In the first step, mRNA was converted to double-stranded cDNA using Superscript Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) and an oligo-dT primer linked to a T7 RNA polymerase binding site sequence (Affymetrix, Santa Clara, CA, USA). Double-stranded cDNA was purified by phenol-chloroform-isoamyl alcohol extraction and a standard ethanol precipitation. In the second step, amplified and labeled cRNA (the target) was produced by a transcription reaction using T7 RNA polymerase and biotin-CTP (Affymetrix). Unincorporated nucleotides were removed using the RNeasy Mini kit (Qiagen) followed by an ethanol precipitation of the labeled target.
Target molecule: cRNA
Hyb protocol: Labeled cRNA targets were chemically fragmented at 95C for 35 min as per Affymetrix’s recommendations. The fragmented material was combined with Affymetrix hybridization controls: biotinylated control oligomer (B2) and biotinylated control cRNAs for BioB, BioC, BioD and CreX (Affymetrix) in hybridization buffer. Following the manufacturer’s recommendations, 6.5 µg of target were hybridized with the GeneChip Human Genome Focus array (Affymetrix) for 16 hours at 45C. Post-hybridization array processing was performed on the Fluidics Station 450 (Affymetrix); array staining and signal amplification were performed according to manufacturer’s recommendations.
|
17A-11H1H_Cfdep_270JR
|
| Sample_geo_accession | GSM335431
| | Sample_status | Public on Jan 30 2009
| | Sample_submission_date | Oct 21 2008
| | Sample_last_update_date | Jan 30 2009
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Unfractionated whole blood collection and RNA isolation were performed using the PAXgene Blood RNA Isolation System (PreAnalytiX, a Qiagen BD Company, Valencia, CA, USA). With the approval of the OHSU Institutional Review Board, blood was collected from healthy donors using eight PAXGene tubes per individual (donor). Four tubes per donor were frozen for subsequent processing and four tubes per donor were processed fresh (see individual sample annotation for storage state details). Following RNA isolation, DNase-treatment was performed as per PAXGene manufacturer’s recommendation. Samples were then pooled within donor and within RNA storage state (fresh/frozen) resulting in two tubes per donor (fresh/frozen). RNA recovery and quality was then assessed by examining UV 260/280 absorbance ratios and RNA size distribution on RNA Nano LabChips (Agilent Technologies, Santa Clara, CA, USA) processed on the Agilent 2100 Bioanalyzer. For inclusion in this study, the RNA mass per donor had to be greater than 12 µg for both fresh and frozen blood aliquots. Samples from three of the four donors (Donors A, C, D) met the quantitative and qualitative criteria and were used in the microarray analysis.
| | Sample_growth_protocol_ch1 | n/a
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | [see characteristics field for details]
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Amplifications and labelings were performed for each donor using one of the following four methods with RNA extracted from fresh and frozen blood samples: Method 1 - Affymetrix one-cycle cDNA synthesis/Affymetrix in vitro-transcription (IVT) on whole blood RNA; Method 2 - Affymetrix one-cycle cDNA/Affymetrix IVT with globin peptide nucleic acids (PNAs) inclusion during cDNA synthesis; and Method 3- RNA pre-treated with Ambion GLOBINclear beads to reduce globin transcripts prior to labeling with the Affymetrix one-cycle cDNA synthesis/Affymetrix IVT. In Method 4- cDNA target was synthesized from whole blood RNA using the NuGEN Ovation Biotin RNA Amplification and Labeling System. See individual sample annotation for protocol details.
| | Sample_hyb_protocol | [see characteristics field for details]
| | Sample_scan_protocol | The distribution of fluorescent material on the processed array was determined using the Affymetrix 3000 GeneArray laser scanner with the 7G upgrade. Image inspection was performed manually immediately following each scan. The array image scan was processed with Affymetrix Microarray Suite, version 5.0, (MAS 5.0) software.
| | Sample_data_processing | Low-level analysis (background correction, normalization, and gene summarization) of microarray data was performed with both Affymetrix Microarray Suite 5.0 (MAS 5.0) and Robust Multi-Array Average (RMA). Individual arrays were analyzed and scaled with MAS 5.0 using manufacturer’s default thresholds for detection calls and with target scaling factor set as 275. For RMA analysis, arrays were normalized in four separate groups based on target labeling method using RMA implemented in Affymetrix Expression Console (version 1.1).
| | Sample_platform_id | GPL201
| | Sample_contact_name | Christina,A,Harrington
| | Sample_contact_email | harringc@ohsu.edu
| | Sample_contact_phone | 503-418-2737
| | Sample_contact_fax | 503-418-9381
| | Sample_contact_laboratory | Affymetrix Microarray Core Facility
| | Sample_contact_department | Gene Microarray Shared Resource
| | Sample_contact_institute | Oregon Health and Science University
| | Sample_contact_address | 3303 SW Bond Ave
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM335nnn/GSM335431/suppl/GSM335431.CEL.gz
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM335nnn/GSM335431/suppl/GSM335431.CHP.gz
| | Sample_series_id | GSE13292
| | Sample_data_row_count | 8793
| | |
|
GSM335432 | GPL201 |
|
D_frozen_Method 3
|
Whole blood, frozen, GLOBINclear_Affymetrix
|
Gender: M
Age: 24, Normal
Biosource: Peripheral whole blood
Donor ID: Donor D
Sample storage state: frozen
Sample protocol: Whole blood was incubated in a PAXGene tube (PreAnalytiX, a Qiagen BD Company, Valencia, CA, USA) for two hours and then transferred to a -80C freezer. RNA was isolated from the frozen PAXGene tube after storage at -80C for at least one week. Treatment protocol: Affymetrix one-cycle cDNA synthesis/Affymetrix IVT with GLOBINclear treated total RNA (GLOBINclear_Affymetrix): Globin mRNA depletion was performed in three steps using the GLOBINclear Kit (Ambion, Austin, TX, USA). First, species-specific biotinylated oligonucleotides complementary to human α- and β-globin mRNA were hybridized with total RNA (5 µg). Second, magnetic streptavidin-coated beads were added to bind the biotinylated-oligonucleotide:globin RNA complexes. Finally, a magnet was used to remove beads with the selectively bound α- and β-globin transcripts. After globin mRNA depletion, RNA quantity was determined by UV absorbance and RNA integrity was assessed using RNA Nano LabChips as described above. RNA recovery following GLOBINclear treatment ranged from 2-5 µg. Label protocol: Affymetrix One-Cycle: Target synthesis was performed following the Affymetrix GeneChip Expression Analysis Technical Manual, rev. 5 (http://www.affymetrix.com/support/technical/manual/expression_manual.affx). Using 1 µg of total RNA as input, messenger RNA was amplified and labeled in two steps. In the first step, mRNA was converted to double-stranded cDNA using Superscript Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) and an oligo-dT primer linked to a T7 RNA polymerase binding site sequence (Affymetrix, Santa Clara, CA, USA). Double-stranded cDNA was purified by phenol-chloroform-isoamyl alcohol extraction and a standard ethanol precipitation. In the second step, amplified and labeled cRNA (the target) was produced by a transcription reaction using T7 RNA polymerase and biotin-CTP (Affymetrix). Unincorporated nucleotides were removed using the RNeasy Mini kit (Qiagen) followed by an ethanol precipitation of the labeled target.
Target molecule: cRNA
Hyb protocol: Labeled cRNA targets were chemically fragmented at 95C for 35 min as per Affymetrix’s recommendations. The fragmented material was combined with Affymetrix hybridization controls: biotinylated control oligomer (B2) and biotinylated control cRNAs for BioB, BioC, BioD and CreX (Affymetrix) in hybridization buffer. Following the manufacturer’s recommendations, 6.5 µg of target were hybridized with the GeneChip Human Genome Focus array (Affymetrix) for 16 hours at 45C. Post-hybridization array processing was performed on the Fluidics Station 450 (Affymetrix); array staining and signal amplification were performed according to manufacturer’s recommendations.
|
18A-11H1H_Dfdep_270JR
|
| Sample_geo_accession | GSM335432
| | Sample_status | Public on Jan 30 2009
| | Sample_submission_date | Oct 21 2008
| | Sample_last_update_date | Jan 30 2009
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Unfractionated whole blood collection and RNA isolation were performed using the PAXgene Blood RNA Isolation System (PreAnalytiX, a Qiagen BD Company, Valencia, CA, USA). With the approval of the OHSU Institutional Review Board, blood was collected from healthy donors using eight PAXGene tubes per individual (donor). Four tubes per donor were frozen for subsequent processing and four tubes per donor were processed fresh (see individual sample annotation for storage state details). Following RNA isolation, DNase-treatment was performed as per PAXGene manufacturer’s recommendation. Samples were then pooled within donor and within RNA storage state (fresh/frozen) resulting in two tubes per donor (fresh/frozen). RNA recovery and quality was then assessed by examining UV 260/280 absorbance ratios and RNA size distribution on RNA Nano LabChips (Agilent Technologies, Santa Clara, CA, USA) processed on the Agilent 2100 Bioanalyzer. For inclusion in this study, the RNA mass per donor had to be greater than 12 µg for both fresh and frozen blood aliquots. Samples from three of the four donors (Donors A, C, D) met the quantitative and qualitative criteria and were used in the microarray analysis.
| | Sample_growth_protocol_ch1 | n/a
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | [see characteristics field for details]
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Amplifications and labelings were performed for each donor using one of the following four methods with RNA extracted from fresh and frozen blood samples: Method 1 - Affymetrix one-cycle cDNA synthesis/Affymetrix in vitro-transcription (IVT) on whole blood RNA; Method 2 - Affymetrix one-cycle cDNA/Affymetrix IVT with globin peptide nucleic acids (PNAs) inclusion during cDNA synthesis; and Method 3- RNA pre-treated with Ambion GLOBINclear beads to reduce globin transcripts prior to labeling with the Affymetrix one-cycle cDNA synthesis/Affymetrix IVT. In Method 4- cDNA target was synthesized from whole blood RNA using the NuGEN Ovation Biotin RNA Amplification and Labeling System. See individual sample annotation for protocol details.
| | Sample_hyb_protocol | [see characteristics field for details]
| | Sample_scan_protocol | The distribution of fluorescent material on the processed array was determined using the Affymetrix 3000 GeneArray laser scanner with the 7G upgrade. Image inspection was performed manually immediately following each scan. The array image scan was processed with Affymetrix Microarray Suite, version 5.0, (MAS 5.0) software.
| | Sample_data_processing | Low-level analysis (background correction, normalization, and gene summarization) of microarray data was performed with both Affymetrix Microarray Suite 5.0 (MAS 5.0) and Robust Multi-Array Average (RMA). Individual arrays were analyzed and scaled with MAS 5.0 using manufacturer’s default thresholds for detection calls and with target scaling factor set as 275. For RMA analysis, arrays were normalized in four separate groups based on target labeling method using RMA implemented in Affymetrix Expression Console (version 1.1).
| | Sample_platform_id | GPL201
| | Sample_contact_name | Christina,A,Harrington
| | Sample_contact_email | harringc@ohsu.edu
| | Sample_contact_phone | 503-418-2737
| | Sample_contact_fax | 503-418-9381
| | Sample_contact_laboratory | Affymetrix Microarray Core Facility
| | Sample_contact_department | Gene Microarray Shared Resource
| | Sample_contact_institute | Oregon Health and Science University
| | Sample_contact_address | 3303 SW Bond Ave
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM335nnn/GSM335432/suppl/GSM335432.CEL.gz
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM335nnn/GSM335432/suppl/GSM335432.CHP.gz
| | Sample_series_id | GSE13292
| | Sample_data_row_count | 8793
| | |
|
GSM335433 | GPL201 |
|
A_fresh_Method 4
|
Whole blood, fresh, no depletion_NuGEN
|
Gender: M
Age: 31, Normal
Biosource: Peripheral whole blood
Donor ID: Donor A
Sample storage state: fresh
Sample protocol: Whole blood was incubated in a PAXGene tube (PreAnalytiX, a Qiagen BD Company, Valencia, CA, USA) for two hours and then RNA was isolated from the fresh sample.
Treatment protocol: NuGEN Ovation Biotin RNA Amplification and Labeling System v.1 (no depletion_NuGEN): No modifications of sample made prior to target synthesis.
Label protocol: NuGEN Ovation Biotin: Ovation labeling and amplification reagents were obtained from NuGEN Technologies, Inc (San Carlos, CA, USA) and biotinylated cDNA targets were prepared according to manufacturer’s instructions. Using a unique, chimeric primer, first strand cDNA was synthesized from 30 ng total RNA. Second strand synthesis of double-stranded cDNA was followed by a linear isothermal amplification (SPIA Amplification™) to produce single-stranded cDNA (the target). A proprietary fragmentation and direct labeling process attached biotin to the amplified target. Target purifications were performed using DNA Clean and Concentrator – 25 (Zymo Research, Orange, CA, USA) and the DyeEx 2.0 Spin Kit (Qiagen).
Target molecule: cDNA
Hyb protocol: Labeled cDNA targets were chemically fragmented at 95C for 35 min as per Affymetrix’s recommendations. The fragmented material was combined with Affymetrix hybridization controls: biotinylated control oligomer (B2) and biotinylated control cRNAs for BioB, BioC, BioD and CreX (Affymetrix) in hybridization buffer. Following the manufacturer’s recommendations, 1.3 µg of target were hybridized with the GeneChip Human Genome Focus array (Affymetrix) for 18 hours at 45C. Post-hybridization array processing was performed on the Fluidics Station 450 (Affymetrix); array staining and signal amplification were performed according to manufacturer’s recommendations.
|
19A-11H1H_Anug_270JR
|
| Sample_geo_accession | GSM335433
| | Sample_status | Public on Jan 30 2009
| | Sample_submission_date | Oct 21 2008
| | Sample_last_update_date | Jan 30 2009
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Unfractionated whole blood collection and RNA isolation were performed using the PAXgene Blood RNA Isolation System (PreAnalytiX, a Qiagen BD Company, Valencia, CA, USA). With the approval of the OHSU Institutional Review Board, blood was collected from healthy donors using eight PAXGene tubes per individual (donor). Four tubes per donor were frozen for subsequent processing and four tubes per donor were processed fresh (see individual sample annotation for storage state details). Following RNA isolation, DNase-treatment was performed as per PAXGene manufacturer’s recommendation. Samples were then pooled within donor and within RNA storage state (fresh/frozen) resulting in two tubes per donor (fresh/frozen). RNA recovery and quality was then assessed by examining UV 260/280 absorbance ratios and RNA size distribution on RNA Nano LabChips (Agilent Technologies, Santa Clara, CA, USA) processed on the Agilent 2100 Bioanalyzer. For inclusion in this study, the RNA mass per donor had to be greater than 12 µg for both fresh and frozen blood aliquots. Samples from three of the four donors (Donors A, C, D) met the quantitative and qualitative criteria and were used in the microarray analysis.
| | Sample_growth_protocol_ch1 | n/a
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | [see characteristics field for details]
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Amplifications and labelings were performed for each donor using one of the following four methods with RNA extracted from fresh and frozen blood samples: Method 1 - Affymetrix one-cycle cDNA synthesis/Affymetrix in vitro-transcription (IVT) on whole blood RNA; Method 2 - Affymetrix one-cycle cDNA/Affymetrix IVT with globin peptide nucleic acids (PNAs) inclusion during cDNA synthesis; and Method 3- RNA pre-treated with Ambion GLOBINclear beads to reduce globin transcripts prior to labeling with the Affymetrix one-cycle cDNA synthesis/Affymetrix IVT. In Method 4- cDNA target was synthesized from whole blood RNA using the NuGEN Ovation Biotin RNA Amplification and Labeling System. See individual sample annotation for protocol details.
| | Sample_hyb_protocol | [see characteristics field for details]
| | Sample_scan_protocol | The distribution of fluorescent material on the processed array was determined using the Affymetrix 3000 GeneArray laser scanner with the 7G upgrade. Image inspection was performed manually immediately following each scan. The array image scan was processed with Affymetrix Microarray Suite, version 5.0, (MAS 5.0) software.
| | Sample_data_processing | Low-level analysis (background correction, normalization, and gene summarization) of microarray data was performed with both Affymetrix Microarray Suite 5.0 (MAS 5.0) and Robust Multi-Array Average (RMA). Individual arrays were analyzed and scaled with MAS 5.0 using manufacturer’s default thresholds for detection calls and with target scaling factor set as 275. For RMA analysis, arrays were normalized in four separate groups based on target labeling method using RMA implemented in Affymetrix Expression Console (version 1.1).
| | Sample_platform_id | GPL201
| | Sample_contact_name | Christina,A,Harrington
| | Sample_contact_email | harringc@ohsu.edu
| | Sample_contact_phone | 503-418-2737
| | Sample_contact_fax | 503-418-9381
| | Sample_contact_laboratory | Affymetrix Microarray Core Facility
| | Sample_contact_department | Gene Microarray Shared Resource
| | Sample_contact_institute | Oregon Health and Science University
| | Sample_contact_address | 3303 SW Bond Ave
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM335nnn/GSM335433/suppl/GSM335433.CEL.gz
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM335nnn/GSM335433/suppl/GSM335433.CHP.gz
| | Sample_series_id | GSE13292
| | Sample_data_row_count | 8793
| | |
|
GSM335434 | GPL201 |
|
C_fresh_Method 4
|
Whole blood, fresh, no depletion_NuGEN
|
Gender: M
Age: 25, Normal
Biosource: Peripheral whole blood
Donor ID: Donor C
Sample storage state: fresh
Sample protocol: Whole blood was incubated in a PAXGene tube (PreAnalytiX, a Qiagen BD Company, Valencia, CA, USA) for two hours and then RNA was isolated from the fresh sample. Treatment protocol: NuGEN Ovation Biotin RNA Amplification and Labeling System v.1 (no depletion_NuGEN): No modifications of sample made prior to target synthesis. Label protocol: NuGEN Ovation Biotin: Ovation labeling and amplification reagents were obtained from NuGEN Technologies, Inc (San Carlos, CA, USA) and biotinylated cDNA targets were prepared according to manufacturer’s instructions. Using a unique, chimeric primer, first strand cDNA was synthesized from 30 ng total RNA. Second strand synthesis of double-stranded cDNA was followed by a linear isothermal amplification (SPIA Amplification™) to produce single-stranded cDNA (the target). A proprietary fragmentation and direct labeling process attached biotin to the amplified target. Target purifications were performed using DNA Clean and Concentrator – 25 (Zymo Research, Orange, CA, USA) and the DyeEx 2.0 Spin Kit (Qiagen).
Target molecule: cDNA
Hyb protocol: Labeled cDNA targets were chemically fragmented at 95C for 35 min as per Affymetrix’s recommendations. The fragmented material was combined with Affymetrix hybridization controls: biotinylated control oligomer (B2) and biotinylated control cRNAs for BioB, BioC, BioD and CreX (Affymetrix) in hybridization buffer. Following the manufacturer’s recommendations, 1.3 µg of target were hybridized with the GeneChip Human Genome Focus array (Affymetrix) for 18 hours at 45C. Post-hybridization array processing was performed on the Fluidics Station 450 (Affymetrix); array staining and signal amplification were performed according to manufacturer’s recommendations.
|
20A-11H1H_Cnug_270JR
|
| Sample_geo_accession | GSM335434
| | Sample_status | Public on Jan 30 2009
| | Sample_submission_date | Oct 21 2008
| | Sample_last_update_date | Jan 30 2009
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Unfractionated whole blood collection and RNA isolation were performed using the PAXgene Blood RNA Isolation System (PreAnalytiX, a Qiagen BD Company, Valencia, CA, USA). With the approval of the OHSU Institutional Review Board, blood was collected from healthy donors using eight PAXGene tubes per individual (donor). Four tubes per donor were frozen for subsequent processing and four tubes per donor were processed fresh (see individual sample annotation for storage state details). Following RNA isolation, DNase-treatment was performed as per PAXGene manufacturer’s recommendation. Samples were then pooled within donor and within RNA storage state (fresh/frozen) resulting in two tubes per donor (fresh/frozen). RNA recovery and quality was then assessed by examining UV 260/280 absorbance ratios and RNA size distribution on RNA Nano LabChips (Agilent Technologies, Santa Clara, CA, USA) processed on the Agilent 2100 Bioanalyzer. For inclusion in this study, the RNA mass per donor had to be greater than 12 µg for both fresh and frozen blood aliquots. Samples from three of the four donors (Donors A, C, D) met the quantitative and qualitative criteria and were used in the microarray analysis.
| | Sample_growth_protocol_ch1 | n/a
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | [see characteristics field for details]
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Amplifications and labelings were performed for each donor using one of the following four methods with RNA extracted from fresh and frozen blood samples: Method 1 - Affymetrix one-cycle cDNA synthesis/Affymetrix in vitro-transcription (IVT) on whole blood RNA; Method 2 - Affymetrix one-cycle cDNA/Affymetrix IVT with globin peptide nucleic acids (PNAs) inclusion during cDNA synthesis; and Method 3- RNA pre-treated with Ambion GLOBINclear beads to reduce globin transcripts prior to labeling with the Affymetrix one-cycle cDNA synthesis/Affymetrix IVT. In Method 4- cDNA target was synthesized from whole blood RNA using the NuGEN Ovation Biotin RNA Amplification and Labeling System. See individual sample annotation for protocol details.
| | Sample_hyb_protocol | [see characteristics field for details]
| | Sample_scan_protocol | The distribution of fluorescent material on the processed array was determined using the Affymetrix 3000 GeneArray laser scanner with the 7G upgrade. Image inspection was performed manually immediately following each scan. The array image scan was processed with Affymetrix Microarray Suite, version 5.0, (MAS 5.0) software.
| | Sample_data_processing | Low-level analysis (background correction, normalization, and gene summarization) of microarray data was performed with both Affymetrix Microarray Suite 5.0 (MAS 5.0) and Robust Multi-Array Average (RMA). Individual arrays were analyzed and scaled with MAS 5.0 using manufacturer’s default thresholds for detection calls and with target scaling factor set as 275. For RMA analysis, arrays were normalized in four separate groups based on target labeling method using RMA implemented in Affymetrix Expression Console (version 1.1).
| | Sample_platform_id | GPL201
| | Sample_contact_name | Christina,A,Harrington
| | Sample_contact_email | harringc@ohsu.edu
| | Sample_contact_phone | 503-418-2737
| | Sample_contact_fax | 503-418-9381
| | Sample_contact_laboratory | Affymetrix Microarray Core Facility
| | Sample_contact_department | Gene Microarray Shared Resource
| | Sample_contact_institute | Oregon Health and Science University
| | Sample_contact_address | 3303 SW Bond Ave
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM335nnn/GSM335434/suppl/GSM335434.CEL.gz
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM335nnn/GSM335434/suppl/GSM335434.CHP.gz
| | Sample_series_id | GSE13292
| | Sample_data_row_count | 8793
| | |
|
GSM335435 | GPL201 |
|
D_fresh_Method 4
|
Whole blood, fresh, no depletion_NuGEN
|
Gender: M
Age: 24, Normal
Biosource: Peripheral whole blood
Donor ID: Donor D
Sample storage state: fresh
Sample protocol: Whole blood was incubated in a PAXGene tube (PreAnalytiX, a Qiagen BD Company, Valencia, CA, USA) for two hours and then RNA was isolated from the fresh sample. Treatment protocol: NuGEN Ovation Biotin RNA Amplification and Labeling System v.1 (no depletion_NuGEN): No modifications of sample made prior to target synthesis. Label protocol: NuGEN Ovation Biotin: Ovation labeling and amplification reagents were obtained from NuGEN Technologies, Inc (San Carlos, CA, USA) and biotinylated cDNA targets were prepared according to manufacturer’s instructions. Using a unique, chimeric primer, first strand cDNA was synthesized from 30 ng total RNA. Second strand synthesis of double-stranded cDNA was followed by a linear isothermal amplification (SPIA Amplification™) to produce single-stranded cDNA (the target). A proprietary fragmentation and direct labeling process attached biotin to the amplified target. Target purifications were performed using DNA Clean and Concentrator – 25 (Zymo Research, Orange, CA, USA) and the DyeEx 2.0 Spin Kit (Qiagen).
Target molecule: cDNA
Hyb protocol: Labeled cDNA targets were chemically fragmented at 95C for 35 min as per Affymetrix’s recommendations. The fragmented material was combined with Affymetrix hybridization controls: biotinylated control oligomer (B2) and biotinylated control cRNAs for BioB, BioC, BioD and CreX (Affymetrix) in hybridization buffer. Following the manufacturer’s recommendations, 1.3 µg of target were hybridized with the GeneChip Human Genome Focus array (Affymetrix) for 18 hours at 45C. Post-hybridization array processing was performed on the Fluidics Station 450 (Affymetrix); array staining and signal amplification were performed according to manufacturer’s recommendations.
|
21A-11H1H_Dnug_270JR
|
| Sample_geo_accession | GSM335435
| | Sample_status | Public on Jan 30 2009
| | Sample_submission_date | Oct 21 2008
| | Sample_last_update_date | Jan 30 2009
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Unfractionated whole blood collection and RNA isolation were performed using the PAXgene Blood RNA Isolation System (PreAnalytiX, a Qiagen BD Company, Valencia, CA, USA). With the approval of the OHSU Institutional Review Board, blood was collected from healthy donors using eight PAXGene tubes per individual (donor). Four tubes per donor were frozen for subsequent processing and four tubes per donor were processed fresh (see individual sample annotation for storage state details). Following RNA isolation, DNase-treatment was performed as per PAXGene manufacturer’s recommendation. Samples were then pooled within donor and within RNA storage state (fresh/frozen) resulting in two tubes per donor (fresh/frozen). RNA recovery and quality was then assessed by examining UV 260/280 absorbance ratios and RNA size distribution on RNA Nano LabChips (Agilent Technologies, Santa Clara, CA, USA) processed on the Agilent 2100 Bioanalyzer. For inclusion in this study, the RNA mass per donor had to be greater than 12 µg for both fresh and frozen blood aliquots. Samples from three of the four donors (Donors A, C, D) met the quantitative and qualitative criteria and were used in the microarray analysis.
| | Sample_growth_protocol_ch1 | n/a
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | [see characteristics field for details]
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Amplifications and labelings were performed for each donor using one of the following four methods with RNA extracted from fresh and frozen blood samples: Method 1 - Affymetrix one-cycle cDNA synthesis/Affymetrix in vitro-transcription (IVT) on whole blood RNA; Method 2 - Affymetrix one-cycle cDNA/Affymetrix IVT with globin peptide nucleic acids (PNAs) inclusion during cDNA synthesis; and Method 3- RNA pre-treated with Ambion GLOBINclear beads to reduce globin transcripts prior to labeling with the Affymetrix one-cycle cDNA synthesis/Affymetrix IVT. In Method 4- cDNA target was synthesized from whole blood RNA using the NuGEN Ovation Biotin RNA Amplification and Labeling System. See individual sample annotation for protocol details.
| | Sample_hyb_protocol | [see characteristics field for details]
| | Sample_scan_protocol | The distribution of fluorescent material on the processed array was determined using the Affymetrix 3000 GeneArray laser scanner with the 7G upgrade. Image inspection was performed manually immediately following each scan. The array image scan was processed with Affymetrix Microarray Suite, version 5.0, (MAS 5.0) software.
| | Sample_data_processing | Low-level analysis (background correction, normalization, and gene summarization) of microarray data was performed with both Affymetrix Microarray Suite 5.0 (MAS 5.0) and Robust Multi-Array Average (RMA). Individual arrays were analyzed and scaled with MAS 5.0 using manufacturer’s default thresholds for detection calls and with target scaling factor set as 275. For RMA analysis, arrays were normalized in four separate groups based on target labeling method using RMA implemented in Affymetrix Expression Console (version 1.1).
| | Sample_platform_id | GPL201
| | Sample_contact_name | Christina,A,Harrington
| | Sample_contact_email | harringc@ohsu.edu
| | Sample_contact_phone | 503-418-2737
| | Sample_contact_fax | 503-418-9381
| | Sample_contact_laboratory | Affymetrix Microarray Core Facility
| | Sample_contact_department | Gene Microarray Shared Resource
| | Sample_contact_institute | Oregon Health and Science University
| | Sample_contact_address | 3303 SW Bond Ave
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM335nnn/GSM335435/suppl/GSM335435.CEL.gz
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM335nnn/GSM335435/suppl/GSM335435.CHP.gz
| | Sample_series_id | GSE13292
| | Sample_data_row_count | 8793
| | |
|
GSM335436 | GPL201 |
|
A_frozen_Method 4
|
Whole blood, frozen, no depletion_NuGEN
|
Gender: M
Age: 31, Normal
Biosource: Peripheral whole blood
Donor ID: Donor A
Sample storage state: frozen
Sample protocol: Whole blood was incubated in a PAXGene tube (PreAnalytiX, a Qiagen BD Company, Valencia, CA, USA) for two hours and then transferred to a -80C freezer. RNA was isolated from the frozen PAXGene tube after storage at -80C for at least one week. Treatment protocol: NuGEN Ovation Biotin RNA Amplification and Labeling System v.1 (no depletion_NuGEN): No modifications of sample made prior to target synthesis. Label protocol: NuGEN Ovation Biotin: Ovation labeling and amplification reagents were obtained from NuGEN Technologies, Inc (San Carlos, CA, USA) and biotinylated cDNA targets were prepared according to manufacturer’s instructions. Using a unique, chimeric primer, first strand cDNA was synthesized from 30 ng total RNA. Second strand synthesis of double-stranded cDNA was followed by a linear isothermal amplification (SPIA Amplification™) to produce single-stranded cDNA (the target). A proprietary fragmentation and direct labeling process attached biotin to the amplified target. Target purifications were performed using DNA Clean and Concentrator – 25 (Zymo Research, Orange, CA, USA) and the DyeEx 2.0 Spin Kit (Qiagen).
Target molecule: cDNA
Hyb protocol: Labeled cDNA targets were chemically fragmented at 95C for 35 min as per Affymetrix’s recommendations. The fragmented material was combined with Affymetrix hybridization controls: biotinylated control oligomer (B2) and biotinylated control cRNAs for BioB, BioC, BioD and CreX (Affymetrix) in hybridization buffer. Following the manufacturer’s recommendations, 1.3 µg of target were hybridized with the GeneChip Human Genome Focus array (Affymetrix) for 18 hours at 45C. Post-hybridization array processing was performed on the Fluidics Station 450 (Affymetrix); array staining and signal amplification were performed according to manufacturer’s recommendations.
|
22A-11H1H_Afnug_270JR
|
| Sample_geo_accession | GSM335436
| | Sample_status | Public on Jan 30 2009
| | Sample_submission_date | Oct 21 2008
| | Sample_last_update_date | Jan 30 2009
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Unfractionated whole blood collection and RNA isolation were performed using the PAXgene Blood RNA Isolation System (PreAnalytiX, a Qiagen BD Company, Valencia, CA, USA). With the approval of the OHSU Institutional Review Board, blood was collected from healthy donors using eight PAXGene tubes per individual (donor). Four tubes per donor were frozen for subsequent processing and four tubes per donor were processed fresh (see individual sample annotation for storage state details). Following RNA isolation, DNase-treatment was performed as per PAXGene manufacturer’s recommendation. Samples were then pooled within donor and within RNA storage state (fresh/frozen) resulting in two tubes per donor (fresh/frozen). RNA recovery and quality was then assessed by examining UV 260/280 absorbance ratios and RNA size distribution on RNA Nano LabChips (Agilent Technologies, Santa Clara, CA, USA) processed on the Agilent 2100 Bioanalyzer. For inclusion in this study, the RNA mass per donor had to be greater than 12 µg for both fresh and frozen blood aliquots. Samples from three of the four donors (Donors A, C, D) met the quantitative and qualitative criteria and were used in the microarray analysis.
| | Sample_growth_protocol_ch1 | n/a
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | [see characteristics field for details]
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Amplifications and labelings were performed for each donor using one of the following four methods with RNA extracted from fresh and frozen blood samples: Method 1 - Affymetrix one-cycle cDNA synthesis/Affymetrix in vitro-transcription (IVT) on whole blood RNA; Method 2 - Affymetrix one-cycle cDNA/Affymetrix IVT with globin peptide nucleic acids (PNAs) inclusion during cDNA synthesis; and Method 3- RNA pre-treated with Ambion GLOBINclear beads to reduce globin transcripts prior to labeling with the Affymetrix one-cycle cDNA synthesis/Affymetrix IVT. In Method 4- cDNA target was synthesized from whole blood RNA using the NuGEN Ovation Biotin RNA Amplification and Labeling System. See individual sample annotation for protocol details.
| | Sample_hyb_protocol | [see characteristics field for details]
| | Sample_scan_protocol | The distribution of fluorescent material on the processed array was determined using the Affymetrix 3000 GeneArray laser scanner with the 7G upgrade. Image inspection was performed manually immediately following each scan. The array image scan was processed with Affymetrix Microarray Suite, version 5.0, (MAS 5.0) software.
| | Sample_data_processing | Low-level analysis (background correction, normalization, and gene summarization) of microarray data was performed with both Affymetrix Microarray Suite 5.0 (MAS 5.0) and Robust Multi-Array Average (RMA). Individual arrays were analyzed and scaled with MAS 5.0 using manufacturer’s default thresholds for detection calls and with target scaling factor set as 275. For RMA analysis, arrays were normalized in four separate groups based on target labeling method using RMA implemented in Affymetrix Expression Console (version 1.1).
| | Sample_platform_id | GPL201
| | Sample_contact_name | Christina,A,Harrington
| | Sample_contact_email | harringc@ohsu.edu
| | Sample_contact_phone | 503-418-2737
| | Sample_contact_fax | 503-418-9381
| | Sample_contact_laboratory | Affymetrix Microarray Core Facility
| | Sample_contact_department | Gene Microarray Shared Resource
| | Sample_contact_institute | Oregon Health and Science University
| | Sample_contact_address | 3303 SW Bond Ave
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM335nnn/GSM335436/suppl/GSM335436.CEL.gz
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM335nnn/GSM335436/suppl/GSM335436.CHP.gz
| | Sample_series_id | GSE13292
| | Sample_data_row_count | 8793
| | |
|
GSM335437 | GPL201 |
|
C_frozen_Method 4
|
Whole blood, frozen, no depletion_NuGEN
|
Gender: M
Age: 25, Normal
Biosource: Peripheral whole blood
Donor ID: Donor C
Sample storage state: frozen
Sample protocol: Whole blood was incubated in a PAXGene tube (PreAnalytiX, a Qiagen BD Company, Valencia, CA, USA) for two hours and then transferred to a -80C freezer. RNA was isolated from the frozen PAXGene tube after storage at -80C for at least one week. Treatment protocol: NuGEN Ovation Biotin RNA Amplification and Labeling System v.1 (no depletion_NuGEN): No modifications of sample made prior to target synthesis. Label protocol: NuGEN Ovation Biotin: Ovation labeling and amplification reagents were obtained from NuGEN Technologies, Inc (San Carlos, CA, USA) and biotinylated cDNA targets were prepared according to manufacturer’s instructions. Using a unique, chimeric primer, first strand cDNA was synthesized from 30 ng total RNA. Second strand synthesis of double-stranded cDNA was followed by a linear isothermal amplification (SPIA Amplification™) to produce single-stranded cDNA (the target). A proprietary fragmentation and direct labeling process attached biotin to the amplified target. Target purifications were performed using DNA Clean and Concentrator – 25 (Zymo Research, Orange, CA, USA) and the DyeEx 2.0 Spin Kit (Qiagen).
Target molecule: cDNA
Hyb protocol: Labeled cDNA targets were chemically fragmented at 95C for 35 min as per Affymetrix’s recommendations. The fragmented material was combined with Affymetrix hybridization controls: biotinylated control oligomer (B2) and biotinylated control cRNAs for BioB, BioC, BioD and CreX (Affymetrix) in hybridization buffer. Following the manufacturer’s recommendations, 1.3 µg of target were hybridized with the GeneChip Human Genome Focus array (Affymetrix) for 18 hours at 45C. Post-hybridization array processing was performed on the Fluidics Station 450 (Affymetrix); array staining and signal amplification were performed according to manufacturer’s recommendations.
|
23A-11H1H_Cfnug_270JR
|
| Sample_geo_accession | GSM335437
| | Sample_status | Public on Jan 30 2009
| | Sample_submission_date | Oct 21 2008
| | Sample_last_update_date | Jan 30 2009
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Unfractionated whole blood collection and RNA isolation were performed using the PAXgene Blood RNA Isolation System (PreAnalytiX, a Qiagen BD Company, Valencia, CA, USA). With the approval of the OHSU Institutional Review Board, blood was collected from healthy donors using eight PAXGene tubes per individual (donor). Four tubes per donor were frozen for subsequent processing and four tubes per donor were processed fresh (see individual sample annotation for storage state details). Following RNA isolation, DNase-treatment was performed as per PAXGene manufacturer’s recommendation. Samples were then pooled within donor and within RNA storage state (fresh/frozen) resulting in two tubes per donor (fresh/frozen). RNA recovery and quality was then assessed by examining UV 260/280 absorbance ratios and RNA size distribution on RNA Nano LabChips (Agilent Technologies, Santa Clara, CA, USA) processed on the Agilent 2100 Bioanalyzer. For inclusion in this study, the RNA mass per donor had to be greater than 12 µg for both fresh and frozen blood aliquots. Samples from three of the four donors (Donors A, C, D) met the quantitative and qualitative criteria and were used in the microarray analysis.
| | Sample_growth_protocol_ch1 | n/a
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | [see characteristics field for details]
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Amplifications and labelings were performed for each donor using one of the following four methods with RNA extracted from fresh and frozen blood samples: Method 1 - Affymetrix one-cycle cDNA synthesis/Affymetrix in vitro-transcription (IVT) on whole blood RNA; Method 2 - Affymetrix one-cycle cDNA/Affymetrix IVT with globin peptide nucleic acids (PNAs) inclusion during cDNA synthesis; and Method 3- RNA pre-treated with Ambion GLOBINclear beads to reduce globin transcripts prior to labeling with the Affymetrix one-cycle cDNA synthesis/Affymetrix IVT. In Method 4- cDNA target was synthesized from whole blood RNA using the NuGEN Ovation Biotin RNA Amplification and Labeling System. See individual sample annotation for protocol details.
| | Sample_hyb_protocol | [see characteristics field for details]
| | Sample_scan_protocol | The distribution of fluorescent material on the processed array was determined using the Affymetrix 3000 GeneArray laser scanner with the 7G upgrade. Image inspection was performed manually immediately following each scan. The array image scan was processed with Affymetrix Microarray Suite, version 5.0, (MAS 5.0) software.
| | Sample_data_processing | Low-level analysis (background correction, normalization, and gene summarization) of microarray data was performed with both Affymetrix Microarray Suite 5.0 (MAS 5.0) and Robust Multi-Array Average (RMA). Individual arrays were analyzed and scaled with MAS 5.0 using manufacturer’s default thresholds for detection calls and with target scaling factor set as 275. For RMA analysis, arrays were normalized in four separate groups based on target labeling method using RMA implemented in Affymetrix Expression Console (version 1.1).
| | Sample_platform_id | GPL201
| | Sample_contact_name | Christina,A,Harrington
| | Sample_contact_email | harringc@ohsu.edu
| | Sample_contact_phone | 503-418-2737
| | Sample_contact_fax | 503-418-9381
| | Sample_contact_laboratory | Affymetrix Microarray Core Facility
| | Sample_contact_department | Gene Microarray Shared Resource
| | Sample_contact_institute | Oregon Health and Science University
| | Sample_contact_address | 3303 SW Bond Ave
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM335nnn/GSM335437/suppl/GSM335437.CEL.gz
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM335nnn/GSM335437/suppl/GSM335437.CHP.gz
| | Sample_series_id | GSE13292
| | Sample_data_row_count | 8793
| | |
|
GSM335438 | GPL201 |
|
D_frozen_Method 4
|
Whole blood, frozen, no depletion_NuGEN
|
Gender: M
Age: 24, Normal
Biosource: Peripheral whole blood
Donor ID: Donor D
Sample storage state: frozen
Sample protocol: Whole blood was incubated in a PAXGene tube (PreAnalytiX, a Qiagen BD Company, Valencia, CA, USA) for two hours and then transferred to a -80C freezer. RNA was isolated from the frozen PAXGene tube after storage at -80C for at least one week. Treatment protocol: NuGEN Ovation Biotin RNA Amplification and Labeling System v.1 (no depletion_NuGEN): No modifications of sample made prior to target synthesis. Label protocol: NuGEN Ovation Biotin: Ovation labeling and amplification reagents were obtained from NuGEN Technologies, Inc (San Carlos, CA, USA) and biotinylated cDNA targets were prepared according to manufacturer’s instructions. Using a unique, chimeric primer, first strand cDNA was synthesized from 30 ng total RNA. Second strand synthesis of double-stranded cDNA was followed by a linear isothermal amplification (SPIA Amplification™) to produce single-stranded cDNA (the target). A proprietary fragmentation and direct labeling process attached biotin to the amplified target. Target purifications were performed using DNA Clean and Concentrator – 25 (Zymo Research, Orange, CA, USA) and the DyeEx 2.0 Spin Kit (Qiagen).
Target molecule: cDNA
Hyb protocol: Labeled cDNA targets were chemically fragmented at 95C for 35 min as per Affymetrix’s recommendations. The fragmented material was combined with Affymetrix hybridization controls: biotinylated control oligomer (B2) and biotinylated control cRNAs for BioB, BioC, BioD and CreX (Affymetrix) in hybridization buffer. Following the manufacturer’s recommendations, 1.3 µg of target were hybridized with the GeneChip Human Genome Focus array (Affymetrix) for 18 hours at 45C. Post-hybridization array processing was performed on the Fluidics Station 450 (Affymetrix); array staining and signal amplification were performed according to manufacturer’s recommendations.
|
24A-11H1H_Dfnug_270JR
|
| Sample_geo_accession | GSM335438
| | Sample_status | Public on Jan 30 2009
| | Sample_submission_date | Oct 21 2008
| | Sample_last_update_date | Jan 30 2009
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Unfractionated whole blood collection and RNA isolation were performed using the PAXgene Blood RNA Isolation System (PreAnalytiX, a Qiagen BD Company, Valencia, CA, USA). With the approval of the OHSU Institutional Review Board, blood was collected from healthy donors using eight PAXGene tubes per individual (donor). Four tubes per donor were frozen for subsequent processing and four tubes per donor were processed fresh (see individual sample annotation for storage state details). Following RNA isolation, DNase-treatment was performed as per PAXGene manufacturer’s recommendation. Samples were then pooled within donor and within RNA storage state (fresh/frozen) resulting in two tubes per donor (fresh/frozen). RNA recovery and quality was then assessed by examining UV 260/280 absorbance ratios and RNA size distribution on RNA Nano LabChips (Agilent Technologies, Santa Clara, CA, USA) processed on the Agilent 2100 Bioanalyzer. For inclusion in this study, the RNA mass per donor had to be greater than 12 µg for both fresh and frozen blood aliquots. Samples from three of the four donors (Donors A, C, D) met the quantitative and qualitative criteria and were used in the microarray analysis.
| | Sample_growth_protocol_ch1 | n/a
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | [see characteristics field for details]
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Amplifications and labelings were performed for each donor using one of the following four methods with RNA extracted from fresh and frozen blood samples: Method 1 - Affymetrix one-cycle cDNA synthesis/Affymetrix in vitro-transcription (IVT) on whole blood RNA; Method 2 - Affymetrix one-cycle cDNA/Affymetrix IVT with globin peptide nucleic acids (PNAs) inclusion during cDNA synthesis; and Method 3- RNA pre-treated with Ambion GLOBINclear beads to reduce globin transcripts prior to labeling with the Affymetrix one-cycle cDNA synthesis/Affymetrix IVT. In Method 4- cDNA target was synthesized from whole blood RNA using the NuGEN Ovation Biotin RNA Amplification and Labeling System. See individual sample annotation for protocol details.
| | Sample_hyb_protocol | [see characteristics field for details]
| | Sample_scan_protocol | The distribution of fluorescent material on the processed array was determined using the Affymetrix 3000 GeneArray laser scanner with the 7G upgrade. Image inspection was performed manually immediately following each scan. The array image scan was processed with Affymetrix Microarray Suite, version 5.0, (MAS 5.0) software.
| | Sample_data_processing | Low-level analysis (background correction, normalization, and gene summarization) of microarray data was performed with both Affymetrix Microarray Suite 5.0 (MAS 5.0) and Robust Multi-Array Average (RMA). Individual arrays were analyzed and scaled with MAS 5.0 using manufacturer’s default thresholds for detection calls and with target scaling factor set as 275. For RMA analysis, arrays were normalized in four separate groups based on target labeling method using RMA implemented in Affymetrix Expression Console (version 1.1).
| | Sample_platform_id | GPL201
| | Sample_contact_name | Christina,A,Harrington
| | Sample_contact_email | harringc@ohsu.edu
| | Sample_contact_phone | 503-418-2737
| | Sample_contact_fax | 503-418-9381
| | Sample_contact_laboratory | Affymetrix Microarray Core Facility
| | Sample_contact_department | Gene Microarray Shared Resource
| | Sample_contact_institute | Oregon Health and Science University
| | Sample_contact_address | 3303 SW Bond Ave
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM335nnn/GSM335438/suppl/GSM335438.CEL.gz
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM335nnn/GSM335438/suppl/GSM335438.CHP.gz
| | Sample_series_id | GSE13292
| | Sample_data_row_count | 8793
| | |
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