|
| Status |
Public on Jun 27, 2008 |
| Title |
Illum_Macrophage_20 |
| Sample type |
RNA |
| |
|
| Source name |
whole blood, macrophage_20
|
| Organism |
Homo sapiens |
| Characteristics |
Age: 65
Gender: male
Tissue: whole blood
Cell type: macrophage
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 |
Refer to the Illumina Gene Expression System Manual
|
| |
|
| Hybridization protocol |
1.5 µg were hybridized to human 6 beadarrays (Illumina, CA, USA) for 16 hours at 55°C. Following hybridization, beadarrays were washed and stained with streptavidin-Cy3 (GE Healthcare, UK).
|
| Scan protocol |
Fluorescent images were obtained with a Beadarray reader and processed with the BeadScan software (Illumina, 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 |
Data were background subtracted using BeadStudio software. (Illumina).
Quality control and pre-processing were performed in the statistical environment R using the Bioconductor packages BeadArray and BeadExplorer Bead-averaged data was normalized using a quantile normalization method Detection scores were calculated in BeadStudio software.
|
| |
|
| Submission date |
Jan 18, 2008 |
| Last update date |
Jun 27, 2008 |
| Contact name |
Francois Cambien |
| E-mail(s) |
francois.cambien@upmc.fr
|
| 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 |
GPL6097 |
| Series (2) |
| GSE10213 |
Performance comparison of Affymetrix and Illumina microarray technologies_IlluminaDataSet |
| GSE11540 |
Performance comparison of Affymetrix and Illumina microarray technologies |
|
