|
| Status |
Public on May 19, 2014 |
| Title |
REH alone_21% Oxygen_s2 |
| Sample type |
RNA |
| |
|
| Source name |
REH alone_21% Oxygen
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: REH
cell type: pre-B acute lymphoblastic leukaemia cells
culture type: cultured alone (monoculture)
|
| Treatment protocol |
All cultures were maintained for 48 hours. To harvest co-cultured cells for analysis, medium and nonattached REH cells were removed and discarded, and attached REH cells and BM-MSC were mobilized with EDTA and collected. Monocultured BM-MSC were similarly harvested, and all cells from REH monocultures were collected. All cell suspensions were stained with anti-human CD90 (Thy-1) antibody as the BM-MSC marker, and anti-human CD45 antibody as the REH cell marker, and then separated with a FACSAria II flow cytometer. Sorted cells were pelleted and lysed with QIAzol lysis reagent for total RNA extraction.
|
| Growth protocol |
BM-MSC isolated from BM of healthy donors, expanded with pooled human platelet lysate and cryopreserved, were thawed and plated for triplicate cultures in RPMI with 10% FBS at 37°C in 5% CO2/air. After 24 hours, medium was replaced with RPMI/FBS alone, for BM-MSC alone, or with RPMI/FBS containing pre-B REH cells for REH/BM-MSC co-cultures, at an REH/BM-MSC ratio of ~2. In parallel, monocultures of REH cells and BM-MSC were similarly prepared and seeded at the same density as co-cultures.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated by using TRIzol reagent (life Technologies), then further purified using the RNeasy plus Mini kit (Qiagen).
|
| Label |
biotin
|
| Label protocol |
200 ng total RNA was amplified and biotin-labeled through a Eberwine procedure using Illumina TotalPrep RNA Amplification kits (Ambion).
|
| |
|
| Hybridization protocol |
As per Illumina instructions, with detection by Streptavidin-Cy3 conjugate.
|
| Scan protocol |
As per instructions for Illumina BeadArray Reader
|
| Description |
5483347151_L
|
| Data processing |
The non-normalized data were generated by GenomeStudio with background correction. The normalized data were generated from bead-level data by methods previously described (PMID: 20805315). In brief, bead-level values were quantile-normalized, adjusted by a model-based background correction method (PMID: 18450815), trimmed by the 3-MAD method employed by Illumina, and then averaged to give a single probe-level value.
|
| |
|
| Submission date |
Mar 03, 2014 |
| Last update date |
May 19, 2014 |
| Contact name |
Wencai Ma |
| Organization name |
MD Anderson Cancer Center
|
| Street address |
7455 Fannin st
|
| City |
Houston |
| ZIP/Postal code |
77054 |
| Country |
USA |
| |
|
| Platform ID |
GPL10558 |
| Series (1) |
| GSE55533 |
Reciprocal leukemia-stroma VCAM-1/VLA-4-dependent activation 1 of NF-κB mediates chemoresistance |
|
