|
| Status |
Public on Aug 31, 2009 |
| Title |
MM_3 |
| Sample type |
RNA |
| |
|
| Source name |
Human bone marrow endothelial cells from Multiple Myeloma patient MM
|
| Organism |
Homo sapiens |
| Characteristics |
Legend: Durie-Salmon Staging System in Multiple Myeloma (Durie BG, Salmon SE Cancer, 1975); immunoglobulin (Ig) protein monoclonal component
Sex: M; stage IIIA; IgA
|
| Treatment protocol |
Centrifugation on Ficoll gradient of heparinized BM-aspirates was followed by polystyrene flask adherence to isolate stromal cells from plasma cells in suspension. Adherent stromal elements were first immunodepleted of macrophages and residual plasma cells with CD14 and CD38 monoclonal antibody (mAb)-coated flasks (mAbs were from Immunotech, Coulter, Marseilles, France), and then incubated with magnetic microbeads coated with Ulex europaeus-1 lectin. The purity and viability of EC cultures grown at least one passage (more than 97% viable cells) were assessed by fluorescence-activated cell sorting (FACS, FACS Canto II, Becton Dickinson, San Jose, CA) with double positivity for factor VIII-related antigen (FVIII-RA, a highly specific EC marker) and CD105 (or endoglyn) as well as for CD14 and CD38 negativity. Analysis of mRNA transcripts for FVIII-RA, CD38, CD105 and IgH VDJ region was also performed by RT-PCR, and EC viability was assessed by trypan blue viable staining.
|
| Growth protocol |
Freshly-isolated ECs were cultured in complete medium RPMI-1640 medium supplemented with 10% heat-inactivated FCS and 1% glutamine to allow cell spreading and growth.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from frozen ECs with TRIzol reagent (Invitrogen, Carlsbad, CA), purified with the RNeasy total RNA Isolation Kit (Qiagen, Valencia, CA).
|
| Label |
biotin
|
| Label protocol |
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 5 micrograms of total RNA (Expression Analysis Technical Manual, Affymetrix).
|
| |
|
| Hybridization protocol |
Following fragmentation, 15 micrograms of cRNA were hybridized for 16 hr and 30 minutes at 45°C on GeneChip HG-U133A Arrays. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.
|
| Scan protocol |
HG-U133A arrays were scanned using the Agilent GeneArray Scanner G2500A (Affymetrix).
|
| Description |
Gene expression profiling data of endothelial cells from Multiple Myeloma patient MM_3
|
| Data processing |
The probe-level signals were converted to expression values using the Bioconductor function for Robust Multi-array Analysis (RMA), in which perfect match intensities are background adjusted, normalized by means of quantile-quantile normalization and log2-transformed.
|
| |
|
| Submission date |
Dec 30, 2008 |
| Last update date |
Sep 01, 2016 |
| Contact name |
Luca Agnelli |
| E-mail(s) |
luca.agnelli@istitutotumori.mi.it, luca.agnelli@gmail.com
|
| Phone |
+390223903581
|
| Organization name |
IRCCS Istituto Nazionale dei Tumori
|
| Department |
Department of Advanced Diagnostics
|
| Street address |
Venezian 1
|
| City |
MILAN |
| ZIP/Postal code |
20133 |
| Country |
Italy |
| |
|
| Platform ID |
GPL96 |
| Series (1) |
| GSE14230 |
Gene expression profiling of bone marrow endothelial cells in patients with multiple myeloma |
|
| Relations |
| Reanalyzed by |
GSE86363 |
