|
| Status |
Public on Jun 27, 2008 |
| Title |
Affy_Monocyte_16 |
| Sample type |
RNA |
| |
|
| Source name |
whole blood, monocyte_16
|
| Organism |
Homo sapiens |
| Characteristics |
Age: 68
Gender: male
Tissue: whole blood
Cell type: monocyte
Patient with symptoms of acute coronary syndrome who had
undergone coronary angiography (who had one stenosis >50 % diagnosed in at least one major coronary artery)
|
| Biomaterial provider |
The department of cardiology of the Pitié-Salpêtrière Hospital, Paris
|
| Treatment protocol |
Blood was drawn under standardized conditions in EDTA tubes: 40 ml samples were collected immediately after coronary angiography and stored at 4°C. Monocytes isolation was performed within 2 hours after Blood drawing. After density gradient centrifugation and washing, PMBC were mixed with CD14 coated beads (MACS, Miltenyi Biotec). To induce phagocytic differentiation, a fraction of the monocytes was incubated for 6 days with macrophage colony stimulating factor (M-CSF, SIGMA).
|
| Growth protocol |
The human monocytes CD14+ obtained after MACS separation were cultured at a concentration of 1x 106 cells/ml in pure RPMI 1640 medium in P6 Petri dishes. After 1h of incubation at 37°C, non adherent cells were removed and the medium was replaced with RPMI 1640 Glu+ medium supplemented with 100U/ml penicillin, 100 µg/ml streptomycin and 10 % FCS, together with 10 ng/ml of macrophage colony stimulating-factor (M-CSF, SIGMA). Cells were incubated for 6 days at 37°C/5% CO2 to induce the phagocytic differentiation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA extraction was done using RNAeasy minikit (Qiagen).
|
| Label |
biotin
|
| Label protocol |
Double stranded cDNA was amplified and labelled using a biotin-NTP mix in the second IVT reaction. The labelled cRNA was then cleaned up and fragmented.
|
| |
|
| Hybridization protocol |
15 µg of cRNA were hybridized to Affymetrix Human U133 plus 2.0 array for 16 hours at 45°C. Following hybridization, the arrays were washed and stained with streptavidin-phycoerythrin.
|
| Scan protocol |
Fluorescent images recorded on a GenChip Scanner 3000 and processed with the GeneChip Operating software (Affymetrix, CA, USA).
|
| Description |
All RNA preparation were checked with Agilent Bioanalyser (RNA 6000 nano-kit) and only RNA with RNA integrity number (RIN) > 8 were accepted for RNA amplification.
|
| Data processing |
Background correction method: Robust Mult-array Average (RMA).
Normalization: quantile
Summarization: for each probe set Median polish method was used to summarized PM intensities into on expression value
Signal intensities were log2-transformed
Detection calls were calculated using the Bioconductor package PANP(Presence-Absence calls with Negative Probesets)
|
| |
|
| Submission date |
Jan 18, 2008 |
| Last update date |
Aug 28, 2018 |
| Contact name |
Francois Cambien |
| E-mail(s) |
[email protected]
|
| Fax |
(33) 140779728
|
| URL |
http://genecanvas.idf.inserm.fr
|
| Organization name |
INSERM
|
| Department |
Cardiovascular Genomics
|
| Lab |
INSERM U937
|
| Street address |
91 Bd de l'Hôpital
|
| City |
Paris |
| State/province |
France |
| ZIP/Postal code |
75634 Paris Cedex 13 |
| Country |
France |
| |
|
| Platform ID |
GPL570 |
| Series (2) |
| GSE11430 |
Performance comparison of Affymetrix and Illumina microarray technologies_AffymetrixDataset |
| GSE11540 |
Performance comparison of Affymetrix and Illumina microarray technologies |
|
| Relations |
| Reanalyzed by |
GSE86362 |
| Reanalyzed by |
GSE119087 |
