|
| Status |
Public on Dec 31, 2010 |
| Title |
Healthy CD4+T cells 11-RP |
| Sample type |
RNA |
| |
|
| Source name |
CD4+T cells
|
| Organism |
Homo sapiens |
| Characteristics |
disease state: Healthy
cell type: CD4+T
twin: R
|
| Treatment protocol |
For gene expression profiling, peripheral blood mononuclear cell (PBMC) were obtained from leukopheresis by density centrifugation over Ficoll-hypaque according to standard procedures. Briefly, heparinized blood was diluted with 1 volume of RPMI medium and gently layered over the Fycoll. Following centrifugation at 660 g for 30 minutes, cells at the interface of the gradient was collected and washed 3 times. Highly purified CD4+ and CD8+ (purity >95%) cell subsets were then sorted by flow cytometry on MoFlo high speed cell sorter (Beckman Coulter, Fullerton, CA) and frozen in Trizol (Invitrogen).
|
| Growth protocol |
none
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol extraction of total RNA was performed according to the manufacturer's instructions.
|
| Label |
Biotin
|
| Label protocol |
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 5 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).
|
| |
|
| Hybridization protocol |
Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 45C on Affymetrix Human Genome U133 Plus 2.0 Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.
|
| Scan protocol |
GeneChips were scanned using the Affymetrix® GeneChip® Scanner 3000.
|
| Description |
n/a
|
| Data processing |
The data were analyzed with Microarray Suite version 5.0 (MAS 5.0) using Affymetrix default analysis settings and global scaling as normalization method. The statistical analysis was performed couple by couple using the Rosetta Resolver® system (Rosetta Biosoftware). The fold-change of each gene was calculated by comparing gene expression in the affected twin with that in the unaffected twin and the genes with the same trend in at least 3 twin pairs out of the 4 studied were selected as candidates to be validated by real-time RT-PCR.
|
| |
|
| Submission date |
Jun 05, 2009 |
| Last update date |
Dec 31, 2010 |
| Contact name |
Viviana Annibali |
| E-mail(s) |
viviana.annibali@uniroma1.it
|
| Phone |
+390633775562
|
| Fax |
+390633775900
|
| Organization name |
University of Rome "Sapienza"
|
| Lab |
DiMA S3
|
| Street address |
Via di Grottarossa,1035
|
| City |
Rome |
| ZIP/Postal code |
00189 |
| Country |
Italy |
| |
|
| Platform ID |
GPL570 |
| Series (1) |
| GSE16461 |
Gene expression profile in CD4+ and CD8+ T cells from identical twins discordant for Multiple sclerosis |
|
