Search results for the GEO ID: GSE21943  |
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GSM ID | GPL ID |
Select for analysis |
Title |
Source name |
Description |
Characteristics |
GSM545847 | GPL96 |
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CD14-negative myeloblasts_MOCK
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Cord blood CD14- myeloblasts, mock
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tissue: cord blood
cell type: CD14- myeloblasts
transfection: mock
time: 48h post-nucleofection
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CD14- MOCK
To achieve an optimal expansion and differentiation of the primary CD34+ hematopoietic cells, a liquid culture was performed by seeding CD34+ cells in six-well plates at a density of 5x105/ml in Iscove’s modified Dulbecco’s medium (IMDM) (GIBCO), added with 20% FCS (Bio-Whittaker), in the presence of human hematopoietic cytokines such as: SCF (50 ng/ml), Flt3-ligand (Flt3-l) (50 ng/ml), IL-6 (10 ng/ml) and IL-3 (10 ng/ml) (all from R&D Systems). After the first week of culture, hematopoietic cells were analyzed by flow cytometry for CD14 antigen expression, estimated at about 15–20% of the entire cell population.
Synthesis of biotinylated cRNA targets, arrays hybridization (Human HG-U133A GeneChip, Affymetrix, Santa Clara,CA), staining and scanning were performed using Affymetrix standard protocols starting from 100ng of total RNA.
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| Sample_geo_accession | GSM545847
| | Sample_status | Public on Jul 14 2010
| | Sample_submission_date | May 20 2010
| | Sample_last_update_date | Jul 14 2010
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | The electroporation of CD14- myeloblasts was performed using the Amaxa Nucleofector® Technology. A mix of three SilencerTM Pre-designed siRNAs targeting human c-Myb was used (Ambion, Inc., The RNA Company®). Each sample was electroporated 3 times, once every 24 hours. For each electroporation, 10^7 CD14- cells were resuspended in 100 µL of Human CD34+ Nucleofection SolutionTM (Amaxa Biosystem), containing 5 µg of siRNA and pulsed with the program X-01. Transfection efficiency was evaluated by the nucleofection of a non-targeting AlexaFluor488-conjugated siRNA (Qiagen) followed by flow cytometric analysis at 6 hours post-transfection. To exclude non-specific effects of siRNA nucleofection, for each experiment, together with the sample transfected with the siRNAs targeting c-Myb (MYBsiRNA), one sample electroporated without siRNAs (MOCK) and one transfected with a non-targeting siRNA (NegCTR) (siCONTROL Non-Targeting Pool, Dharmacon) were performed. After each transfection, myeloblasts were plated (10^6/ml) into prewarmed IMDM, added with 20% FCS and human cytokines, as SCF (10 ng/ml), Flt3L (10 ng/ml), IL-6 (10 ng/ml) and IL-3 (10 ng/ml), in 6-well plates. Cells were analyzed 24 and 48 hours post-nucleofection for both cell viability and c-Myb expression.
| | Sample_growth_protocol_ch1 | Human CD14- myeloblasts were obtained by Cord Blood CD34+ stem/progenitor cells liquid culture according to Montanari M et al. (Montanari M, Gemelli C, Tenedini E, et al. Cell Death Differ. 2005;12:1588-1600) and purified using magnetic cell sorting procedure (EasySep Human CD14 Positive Selection kit, StemCell Technologies Inc). After immunomagnetic separation, CD14- cells were seeded in six-well plates at a density of 106/ml in IMDM (GIBCO, Grand Island), added with 20% Fetal Calf Serum (FCS, Bio-Whittaker), in the presence of human hematopoietic cytokines, namely SCF (10 ng/ml), Flt3L (10 ng/ml), IL-6 (10 ng/ml) and IL-3 (10 ng/ml) (all from R&D Systems) and electroporated 24 hours later. The purity of CD14- cells, assessed by flow cytometry, was always >95%.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | RNAs originating from 3 different experiments were pooled in order to obtain at least 100ng per sample. Total cellular RNA was extracted from 3x10^5 cells of each sample using RNeasy Micro kit (Qiagen) following the protocol supplied by the manufacturer. Disposable RNA chips (Agilent RNA 6000 Nano LabChip kit, Agilent Technologies) were used to determine the purity and integrity of RNA samples using Agilent 2100 Bioanalyzer. NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies) was used to evaluate the RNA sample concentration, and 260/280 and 260/230 nm ratios were considered to verify the purity of total RNA.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Total cellular RNA pools (100 ng) of MOCK, NegCTR and MYBsiRNA CD34+ cells, obtained from three independent experiments, were converted in biotinylated cRNA according to the two-cycle target labeling protocol advised by Affymetrix (Affymetrix, Santa Clara,CA). Labeled cRNA was purified using Affymetrix GeneChip Sample Cleanup Module and fragmented (15 µg) as described in the Affymetrix GeneChip protocol. Disposable RNA chips (Agilent RNA 6000 Nano LabChip kit, Agilent Technologies, Waldbrunn, Germany) and Agilent 2100 Bioanalyzer were used to determine the cRNA concentration/quality as well as to optimize the cRNA fragmentation. NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies) was used to evaluate the cRNA sample concentration.
| | Sample_hyb_protocol | The fragmented cRNA was then hybridized to an identical lot of Affymetrix Human HG-U133A GeneChip arrays for 16 hours. GeneChips were washed and stained using the instrument’s standard Eukaryotic GE WS2v4 protocol, using antibody-mediated signal amplification.
| | Sample_scan_protocol | GeneChips were scanned using the Affymetrix GeneChip scanner 3000, enabled for High-Resolution Scanning.
| | Sample_data_processing | The data were analyzed with GeneChip-Operating-Software (GCOS) using Affymetrix default analysis settings and global scaling as normalization method (Scaling to TGT value=400). The GCOS absolute analysis algorithm was used to determine the mRNA expression levels (Signal), while the GCOS comparison analysis algorithm was used in order to compare gene expression levels between two samples.
| | Sample_platform_id | GPL96
| | Sample_contact_name | Rossella,,Manfredini
| | Sample_contact_email | manfredini.rossella@unimo.it
| | Sample_contact_phone | +390592058065
| | Sample_contact_fax | +390592058115
| | Sample_contact_department | Life Sciences
| | Sample_contact_institute | Centre for Regenerative Medicine
| | Sample_contact_address | Via Gottardi 100
| | Sample_contact_city | Modena
| | Sample_contact_zip/postal_code | 41100
| | Sample_contact_country | Italy
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM545nnn/GSM545847/suppl/GSM545847_CD14-_MOCK.CEL.gz
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM545nnn/GSM545847/suppl/GSM545847_CD14-_MOCK.CHP.gz
| | Sample_series_id | GSE21943
| | Sample_data_row_count | 22283
| | |
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GSM545848 | GPL96 |
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CD14-negative myeloblasts_NegCTR
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Cord blood CD14- myeloblasts, negative control siRNA
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tissue: cord blood
cell type: CD14- myeloblasts
transfection: non-targeting siRNA
time: 48h post-nucleofection
|
CD14- NegCTR
To achieve an optimal expansion and differentiation of the primary CD34+ hematopoietic cells, a liquid culture was performed by seeding CD34+ cells in six-well plates at a density of 5x105/ml in Iscove’s modified Dulbecco’s medium (IMDM) (GIBCO), added with 20% FCS (Bio-Whittaker), in the presence of human hematopoietic cytokines such as: SCF (50 ng/ml), Flt3-ligand (Flt3-l) (50 ng/ml), IL-6 (10 ng/ml) and IL-3 (10 ng/ml) (all from R&D Systems). After the first week of culture, hematopoietic cells were analyzed by flow cytometry for CD14 antigen expression, estimated at about 15–20% of the entire cell population.
Synthesis of biotinylated cRNA targets, arrays hybridization (Human HG-U133A GeneChip, Affymetrix, Santa Clara,CA), staining and scanning were performed using Affymetrix standard protocols starting from 100ng of total RNA.
|
| Sample_geo_accession | GSM545848
| | Sample_status | Public on Jul 14 2010
| | Sample_submission_date | May 20 2010
| | Sample_last_update_date | Jul 14 2010
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | The electroporation of CD14- myeloblasts was performed using the Amaxa Nucleofector® Technology. A mix of three SilencerTM Pre-designed siRNAs targeting human c-Myb was used (Ambion, Inc., The RNA Company®). Each sample was electroporated 3 times, once every 24 hours. For each electroporation, 10^7 CD14- cells were resuspended in 100 µL of Human CD34+ Nucleofection SolutionTM (Amaxa Biosystem), containing 5 µg of siRNA and pulsed with the program X-01. Transfection efficiency was evaluated by the nucleofection of a non-targeting AlexaFluor488-conjugated siRNA (Qiagen) followed by flow cytometric analysis at 6 hours post-transfection. To exclude non-specific effects of siRNA nucleofection, for each experiment, together with the sample transfected with the siRNAs targeting c-Myb (MYBsiRNA), one sample electroporated without siRNAs (MOCK) and one transfected with a non-targeting siRNA (NegCTR) (siCONTROL Non-Targeting Pool, Dharmacon) were performed. After each transfection, myeloblasts were plated (10^6/ml) into prewarmed IMDM, added with 20% FCS and human cytokines, as SCF (10 ng/ml), Flt3L (10 ng/ml), IL-6 (10 ng/ml) and IL-3 (10 ng/ml), in 6-well plates. Cells were analyzed 24 and 48 hours post-nucleofection for both cell viability and c-Myb expression.
| | Sample_growth_protocol_ch1 | Human CD14- myeloblasts were obtained by Cord Blood CD34+ stem/progenitor cells liquid culture according to Montanari M et al. (Montanari M, Gemelli C, Tenedini E, et al. Cell Death Differ. 2005;12:1588-1600) and purified using magnetic cell sorting procedure (EasySep Human CD14 Positive Selection kit, StemCell Technologies Inc). After immunomagnetic separation, CD14- cells were seeded in six-well plates at a density of 106/ml in IMDM (GIBCO, Grand Island), added with 20% Fetal Calf Serum (FCS, Bio-Whittaker), in the presence of human hematopoietic cytokines, namely SCF (10 ng/ml), Flt3L (10 ng/ml), IL-6 (10 ng/ml) and IL-3 (10 ng/ml) (all from R&D Systems) and electroporated 24 hours later. The purity of CD14- cells, assessed by flow cytometry, was always >95%.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | RNAs originating from 3 different experiments were pooled in order to obtain at least 100ng per sample. Total cellular RNA was extracted from 3x10^5 cells of each sample using RNeasy Micro kit (Qiagen) following the protocol supplied by the manufacturer. Disposable RNA chips (Agilent RNA 6000 Nano LabChip kit, Agilent Technologies) were used to determine the purity and integrity of RNA samples using Agilent 2100 Bioanalyzer. NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies) was used to evaluate the RNA sample concentration, and 260/280 and 260/230 nm ratios were considered to verify the purity of total RNA.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Total cellular RNA pools (100 ng) of MOCK, NegCTR and MYBsiRNA CD34+ cells, obtained from three independent experiments, were converted in biotinylated cRNA according to the two-cycle target labeling protocol advised by Affymetrix (Affymetrix, Santa Clara,CA). Labeled cRNA was purified using Affymetrix GeneChip Sample Cleanup Module and fragmented (15 µg) as described in the Affymetrix GeneChip protocol. Disposable RNA chips (Agilent RNA 6000 Nano LabChip kit, Agilent Technologies, Waldbrunn, Germany) and Agilent 2100 Bioanalyzer were used to determine the cRNA concentration/quality as well as to optimize the cRNA fragmentation. NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies) was used to evaluate the cRNA sample concentration.
| | Sample_hyb_protocol | The fragmented cRNA was then hybridized to an identical lot of Affymetrix Human HG-U133A GeneChip arrays for 16 hours. GeneChips were washed and stained using the instrument’s standard Eukaryotic GE WS2v4 protocol, using antibody-mediated signal amplification.
| | Sample_scan_protocol | GeneChips were scanned using the Affymetrix GeneChip scanner 3000, enabled for High-Resolution Scanning.
| | Sample_data_processing | The data were analyzed with GeneChip-Operating-Software (GCOS) using Affymetrix default analysis settings and global scaling as normalization method (Scaling to TGT value=400). The GCOS absolute analysis algorithm was used to determine the mRNA expression levels (Signal), while the GCOS comparison analysis algorithm was used in order to compare gene expression levels between two samples.
| | Sample_platform_id | GPL96
| | Sample_contact_name | Rossella,,Manfredini
| | Sample_contact_email | manfredini.rossella@unimo.it
| | Sample_contact_phone | +390592058065
| | Sample_contact_fax | +390592058115
| | Sample_contact_department | Life Sciences
| | Sample_contact_institute | Centre for Regenerative Medicine
| | Sample_contact_address | Via Gottardi 100
| | Sample_contact_city | Modena
| | Sample_contact_zip/postal_code | 41100
| | Sample_contact_country | Italy
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM545nnn/GSM545848/suppl/GSM545848_CD14-_NegCTR.CEL.gz
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM545nnn/GSM545848/suppl/GSM545848_CD14-_NegCTR.CHP.gz
| | Sample_series_id | GSE21943
| | Sample_data_row_count | 22283
| | |
|
GSM545849 | GPL96 |
|
CD14-negative myeloblasts_MYBsiRNA
|
Cord blood CD14- myeloblasts, Myb siRNA
|
tissue: cord blood
cell type: CD14- myeloblasts
transfection: Myb siRNA
time: 48h post-nucleofection
|
CD14- MYBsiRNA
To achieve an optimal expansion and differentiation of the primary CD34+ hematopoietic cells, a liquid culture was performed by seeding CD34+ cells in six-well plates at a density of 5x105/ml in Iscove’s modified Dulbecco’s medium (IMDM) (GIBCO), added with 20% FCS (Bio-Whittaker), in the presence of human hematopoietic cytokines such as: SCF (50 ng/ml), Flt3-ligand (Flt3-l) (50 ng/ml), IL-6 (10 ng/ml) and IL-3 (10 ng/ml) (all from R&D Systems). After the first week of culture, hematopoietic cells were analyzed by flow cytometry for CD14 antigen expression, estimated at about 15–20% of the entire cell population.
Synthesis of biotinylated cRNA targets, arrays hybridization (Human HG-U133A GeneChip, Affymetrix, Santa Clara,CA), staining and scanning were performed using Affymetrix standard protocols starting from 100ng of total RNA.
|
| Sample_geo_accession | GSM545849
| | Sample_status | Public on Jul 14 2010
| | Sample_submission_date | May 20 2010
| | Sample_last_update_date | Jul 14 2010
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | The electroporation of CD14- myeloblasts was performed using the Amaxa Nucleofector® Technology. A mix of three SilencerTM Pre-designed siRNAs targeting human c-Myb was used (Ambion, Inc., The RNA Company®). Each sample was electroporated 3 times, once every 24 hours. For each electroporation, 10^7 CD14- cells were resuspended in 100 µL of Human CD34+ Nucleofection SolutionTM (Amaxa Biosystem), containing 5 µg of siRNA and pulsed with the program X-01. Transfection efficiency was evaluated by the nucleofection of a non-targeting AlexaFluor488-conjugated siRNA (Qiagen) followed by flow cytometric analysis at 6 hours post-transfection. To exclude non-specific effects of siRNA nucleofection, for each experiment, together with the sample transfected with the siRNAs targeting c-Myb (MYBsiRNA), one sample electroporated without siRNAs (MOCK) and one transfected with a non-targeting siRNA (NegCTR) (siCONTROL Non-Targeting Pool, Dharmacon) were performed. After each transfection, myeloblasts were plated (10^6/ml) into prewarmed IMDM, added with 20% FCS and human cytokines, as SCF (10 ng/ml), Flt3L (10 ng/ml), IL-6 (10 ng/ml) and IL-3 (10 ng/ml), in 6-well plates. Cells were analyzed 24 and 48 hours post-nucleofection for both cell viability and c-Myb expression.
| | Sample_growth_protocol_ch1 | Human CD14- myeloblasts were obtained by Cord Blood CD34+ stem/progenitor cells liquid culture according to Montanari M et al. (Montanari M, Gemelli C, Tenedini E, et al. Cell Death Differ. 2005;12:1588-1600) and purified using magnetic cell sorting procedure (EasySep Human CD14 Positive Selection kit, StemCell Technologies Inc). After immunomagnetic separation, CD14- cells were seeded in six-well plates at a density of 106/ml in IMDM (GIBCO, Grand Island), added with 20% Fetal Calf Serum (FCS, Bio-Whittaker), in the presence of human hematopoietic cytokines, namely SCF (10 ng/ml), Flt3L (10 ng/ml), IL-6 (10 ng/ml) and IL-3 (10 ng/ml) (all from R&D Systems) and electroporated 24 hours later. The purity of CD14- cells, assessed by flow cytometry, was always >95%.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | RNAs originating from 3 different experiments were pooled in order to obtain at least 100ng per sample. Total cellular RNA was extracted from 3x10^5 cells of each sample using RNeasy Micro kit (Qiagen) following the protocol supplied by the manufacturer. Disposable RNA chips (Agilent RNA 6000 Nano LabChip kit, Agilent Technologies) were used to determine the purity and integrity of RNA samples using Agilent 2100 Bioanalyzer. NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies) was used to evaluate the RNA sample concentration, and 260/280 and 260/230 nm ratios were considered to verify the purity of total RNA.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Total cellular RNA pools (100 ng) of MOCK, NegCTR and MYBsiRNA CD34+ cells, obtained from three independent experiments, were converted in biotinylated cRNA according to the two-cycle target labeling protocol advised by Affymetrix (Affymetrix, Santa Clara,CA). Labeled cRNA was purified using Affymetrix GeneChip Sample Cleanup Module and fragmented (15 µg) as described in the Affymetrix GeneChip protocol. Disposable RNA chips (Agilent RNA 6000 Nano LabChip kit, Agilent Technologies, Waldbrunn, Germany) and Agilent 2100 Bioanalyzer were used to determine the cRNA concentration/quality as well as to optimize the cRNA fragmentation. NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies) was used to evaluate the cRNA sample concentration.
| | Sample_hyb_protocol | The fragmented cRNA was then hybridized to an identical lot of Affymetrix Human HG-U133A GeneChip arrays for 16 hours. GeneChips were washed and stained using the instrument’s standard Eukaryotic GE WS2v4 protocol, using antibody-mediated signal amplification.
| | Sample_scan_protocol | GeneChips were scanned using the Affymetrix GeneChip scanner 3000, enabled for High-Resolution Scanning.
| | Sample_data_processing | The data were analyzed with GeneChip-Operating-Software (GCOS) using Affymetrix default analysis settings and global scaling as normalization method (Scaling to TGT value=400). The GCOS absolute analysis algorithm was used to determine the mRNA expression levels (Signal), while the GCOS comparison analysis algorithm was used in order to compare gene expression levels between two samples.
| | Sample_platform_id | GPL96
| | Sample_contact_name | Rossella,,Manfredini
| | Sample_contact_email | manfredini.rossella@unimo.it
| | Sample_contact_phone | +390592058065
| | Sample_contact_fax | +390592058115
| | Sample_contact_department | Life Sciences
| | Sample_contact_institute | Centre for Regenerative Medicine
| | Sample_contact_address | Via Gottardi 100
| | Sample_contact_city | Modena
| | Sample_contact_zip/postal_code | 41100
| | Sample_contact_country | Italy
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM545nnn/GSM545849/suppl/GSM545849_CD14-_MYBsiRNA.CEL.gz
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM545nnn/GSM545849/suppl/GSM545849_CD14-_MYBsiRNA.CHP.gz
| | Sample_series_id | GSE21943
| | Sample_data_row_count | 22283
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