|
| Status |
Public on Mar 21, 2013 |
| Title |
Multiple Myeloma Patient 4 |
| Sample type |
RNA |
| |
|
| Source name |
bone-marrow mesenchymal stromal cells extracted from myeloma patient
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: bone marrow mesenchymal stromal cells (BM-MSCs)
disease state: multiple myeloma
mm stage: IIIa
Sex: F
age: 56
|
| Growth protocol |
Bone marrow was harvested from the sternum or iliac crest of patients after informed consent. Mononuclear cells were isolated by density gradient centrifugation (LinfoSep, BiomediCs, Madrid, Spain), washed in Hank’s buffered salt solution (HBSS, Lonza Europe, Verviers, Belgium) and seeded at 20,000 cells/cm2 in Dulbecco’s Modified Eagle’s Medium-low glucose (DMEM-LG, Lonza) supplemented with 15% of fetal bovine serum (FBS) (Sigma-Aldrich, Bornem, Belgium), 2 mM of L-glutamine, 50 U/ml of penicillin, and 50 µg/ml of streptomycin (all from Lonza). Cells were incubated at 37°C in a 5% CO2 and 95% humidified atmosphere.
After 48 hours, nonadherent cells were removed by washing and the medium was changed once a week. When subconfluency (80–90%) was achieved, adherent cells were detached with TrypLE Select solution (Lonza) and expanded by replating at a lower density (± 600 cells/cm2).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from each cell culture was extracted in a single step by the TriPure Isolation Reagent (Roche Applied Science, Vilvoorde, Belgium). We performed the reverse transcription reaction with 2 µg RNA using qScript cDNA SuperMix (Quanta Bioscienes, Gaithersburg, MD, USA).
|
| Label |
Biotin
|
| Label protocol |
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 1.5 µg total RNA (www.affymetrix.com).
|
| |
|
| Hybridization protocol |
Following fragmentation, 16 µg of cRNA were hybridized for 16 hr at 45C on Affymetrix GeneChip® Human Genome U133 Plus 2.0 Array. GeneChips were washed and stained in the Affymetrix GeneChip Fluidics Station 450 according to the Affymetrix protocol (www.affymetrix.com).
|
| Scan protocol |
GeneChips were scanned using the Affymetrix GeneChip Scanner 3000 7G.
|
| Description |
MM4
Biological rep4.
|
| Data processing |
Image analysis and probe quantification were done with Affymetrix software, which produced raw probe intensity data in the Affymetrix CEL files. Normalization was done with the RMA method, which processed a group of CEL files simultaneously using BRB arrays tools.
|
| |
|
| Submission date |
Mar 13, 2012 |
| Last update date |
Mar 21, 2013 |
| Contact name |
Thibaud André |
| E-mail(s) |
[email protected]
|
| Organization name |
ULB - Jules Bordet Insitute
|
| Lab |
Laboratory of Clinical Cell Therapy
|
| Street address |
Boulevard de Waterloo, 121
|
| City |
Brussels |
| ZIP/Postal code |
1000 |
| Country |
Belgium |
| |
|
| Platform ID |
GPL570 |
| Series (1) |
| GSE36474 |
Comparison of bone-marrow mesenchymal stromal cells from multiple myeloma patients and healthy donors |
|
