|
| Status |
Public on Jul 29, 2009 |
| Title |
CD4+ T-cells_healty control_donor3 |
| Sample type |
RNA |
| |
|
| Source name |
CD4+ T-cells purified with immunomagnetic beads from the pheripheral blood lymphocytes of the healthy control C3
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: peripheral blood
cell type: CD4+ T-cells
treatment type: normal donor
|
| Treatment protocol |
Mononuclear cells from peripheral blood were isolated by density gradient centrifugation on Ficoll-Hypaque (Nycomed Pharma A/S). Thereafter, the CD4 and CD8 T-cell subsets were purified with positive selection by immunomagnetic technique using anti-CD4 and anti-CD8 antibody-coated microbeads (Miltenyi Biotec). Two steps of purification were performed to increase the purity to greater than 98%-99%. RNA was directly extracted from cell pellets.
|
| Growth protocol |
Heparinized blood samples were obtained from patients with ADA-SCID and age-matched healthy controls after parents of patients gave informed consent following standard ethical procedures and with the approval of the Institutional Ethical Committee and immediately processed for mononuclear cell purification.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated from 1x10^6 cells using Eurozol reagent (Euroclone S.p.A., Italy) according to the manufacturer's instructions. Disposable RNA chips (Agilent RNA 6000 Nano LabChip kit, Agilent Technologies, Waldbrunn, Germany) were used to determine the concentration, purity, and integrity of RNA samples using Agilent 2100 Bioanalyzer.
|
| Label |
biotin
|
| Label protocol |
The one-cycle target labeling assay was performed using Affymetrix standard protocols (Affymetrix, Santa Clara,CA). Briefly, biotin-labeled target synthesis was performed, starting from 5 µg of total cellular RNA, according to the protocol supplied by the manufacturer (Affymetrix, Santa Clara, CA). Labeled cRNA was purified using Affymetrix GeneChip Sample Cleanup Module and fragmented (15 µg) as described in the Affymetrix GeneChip protocol. Disposable RNA chips (Agilent RNA 6000 Nano LabChip kit, Agilent Technologies, Waldbrunn, Germany) and Agilent 2100 Bioanalyzer were used to determine the cRNA concentration, quality and to optimize the cRNA fragmentation.
|
| |
|
| Hybridization protocol |
Affymetrix Human HG-U133A GeneChip array hybridization and staining was performed using Affymetrix standard protocols (Affymetrix, Santa Clara,CA). The fragmented cRNA were hybridized to an identical lot of Affymetrix Human HG-U133A GeneChip arrays for 16 hours. GeneChips were washed and stained using the instrument’s standard Eukaryotic GE WS2v4 protocol, using antibody-mediated signal amplification.
|
| Scan protocol |
GeneChips were scanned using the Affymetrix GeneChip scanner 3000 enabled for High-Resolution Scanning.
|
| Description |
Gene expression data from CD4+ T-cells isolated from healty control
|
| Data processing |
The data were analyzed with Microarray Suite version 5.0 (MAS 5.0) using Affymetrix default analysis settings. Data analysis was performed using GeneSpring® GX 7.3.1 version (Agilent Technologies).
|
| |
|
| Submission date |
Jul 27, 2009 |
| Last update date |
Sep 01, 2016 |
| Contact name |
Alessandro Aiuti |
| E-mail(s) |
a.aiuti@hsr.it
|
| Phone |
+39-02-26434435
|
| Fax |
+39-02-26434668
|
| Organization name |
San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET)
|
| Street address |
via olgettina 58
|
| City |
Milan |
| ZIP/Postal code |
20132 |
| Country |
Italy |
| |
|
| Platform ID |
GPL96 |
| Series (1) |
| GSE17354 |
Gene expression profiling of CD4+ and CD8+ T-cells from gene therapy treated ADA patients and from healthy controls |
|
| Relations |
| Reanalyzed by |
GSE86363 |
