|
| Status |
Public on Aug 31, 2008 |
| Title |
RNG_Macrophage_Pool33 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Human macrophage labeled with Cy5
|
| Organism |
Homo sapiens |
| Characteristics |
Age: 57
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 |
For the isolation of PMBC from anti-coagulated blood and removal of dead cells, a density gradient centrifugation using Ficoll–Paque (GE Healthcare) gradient was used. PMBC were washed with PBS and the supernatant completely removed. The cell pellet was re-suspended in 80 µl of PBS/BSA buffer per 107 total cells and 10 µl of MACS CD14 (Miltenyi Biotec) were added carefully, mixed and incubated for 15 min at 6°-12°C. The cells were washed by adding 10-20X labelling volume of buffer, centrifuged at 300 g for 10 min. The cells were re-suspended in 500 µl of buffer before magnetic separation as described by the manufacturer.
|
| 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 was extracted from whole blood using RNeasy mini kit of Qiagen.
|
| Label |
Cy5
|
| Label protocol |
RNA was amplified and labeled with Cy5
|
| |
|
| Channel 2 |
| Source name |
Pool of 50 different monocyte and macrophage samples
|
| Organism |
Homo sapiens |
| Characteristics |
Patients 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 |
1µg total RNA from 50 different samples were mixed, then 750ng mixed total RNA was amplified and labelled with Cy3
|
| 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 was extracted from whole blood using RNeasy mini kit of Qiagen according to the manufacturer’s instructions.
|
| Label |
Cy3
|
| Label protocol |
RNA was amplified and labeled with Cy3
|
| |
|
| |
| Hybridization protocol |
Hybridizations were performed using the Agilent in situ hybridization kit. For each sample, 750 ng of Cy5-labeled, linearly amplified cRNA was mixed with equal amounts of Cy3-labelled, linearly amplified reference cRNA. The mixture was then fragmented by incubation with the fragmentation buffer at 60°C for 30 minutes. An equal amount of 2x hybridization buffer was added to the fragmented cRNA mixture and hybridized to whole-genome oligo arrays (Human-25K, Réseau National des Génopoles, France) at 60°C for 17 hours
|
| Scan protocol |
Fluorescent images of hybridized microarrays were obtained with a GenePix 4000 Axon Instruments scanner and provessed with the GenePix software (Axon Instruments).
|
| 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 |
The Bioconductor packages marray and Limma were used to perform quality control and preprocessing.
Background correction method: normexp
Within array print-tip loess normalization was performed for each spot followed by between array quantile normalization.
bad spots were not included in the analysis.
|
| |
|
| Submission date |
Jan 21, 2008 |
| Last update date |
Jul 28, 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 |
GPL1946 |
| Series (1) |
| GSE10220 |
Gene expression profiling of human monocytes and monocyte-derived-macrophages |
|
