|
| Status |
Public on Feb 22, 2008 |
| Title |
Recent-onset T1D_sample3 |
| Sample type |
RNA |
| |
|
| Source name |
Un-stimulated CD4+CD25+high regulatory T cells from PBMCs
|
| Organism |
Homo sapiens |
| Characteristics |
HLA risk: Medium
Height: 166.3 cm
Weight: 64.5 kg
BMI: 23.2
Age: 18.16 years
Gender: Female
Race: Other
Glucose: 114 mg/dl
HbA1c: 7.4%
|
| Treatment protocol |
The PBMCs were counted and stained with a cocktail of fluorochrome-conjugated monoclonal antibodies in PBS (APC-aCD4 (clone RPA-T4), APC-Cy7-aCD25 (clone M-A251), FITC-aCD14 (clone M5E2), FITC-aCD32 (clone FL18.26), FITC-aCD116 (cloneM5D12), PE-Cy7-aCD8 (clone RPA-T8) all from BD Pharmingen, San Diego, CA) and sorted on a FACSAria (BD Biosciences, San Diego, CA). Cells were first gated on the live lymphocyte population, eliminating dead cells and debris. Two additional gates were set up to eliminate non-CD4 T cells (FITC-stained cells and CD8-PE-Cy7 stained cells). CD4+ T cells were further gated as CD4+25- and CD4+CD25+high using the Fluorochrome Minus One (FMO) method, which allows for a more precise definition of cells having fluorescence above the background level. The 1% of cells expressing the highest level of CD25 were collected and defined as CD4+25+high T cells, known to be enriched for Tregs.
|
| Growth protocol |
Peripheral blood mononuclear cells (PBMCs) were collected using vacutainers with ACD solution B of trisodium citrate and isolated using Ficoll-Hypaque density centrifugation according to the recommended protocol (Amersham Pharmacia, Uppsala, Sweden).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from T cells using TRIzol Reagent (Invitrogen) according to the recommended protocol. The GeneChip® Human Genome U133 Plus 2.0 array was selected for this study which interrogates more than 47,000 probe sets, representing roughly 39,000 unique UniGenes.
|
| Label |
biotin
|
| Label protocol |
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 6 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).
|
| |
|
| Hybridization protocol |
Purified total RNA (~50ng) was amplified using the two-cycle cDNA synthesis kit (Affymetrix, 900432) and cRNA was synthesized, labeled, fragmented and hybridized to the arrays in accordance to standard Affymetrix protocols (Affymetrix, Santa Clara, CA).
|
| Scan protocol |
After hybridization arrays were washed, stained with PE-conjugated streptavidin (Molecular Probes, Eugene, OR) and scanned on a GeneChip® Scanner 3000.
|
| Description |
Gene expression data from unstimulated Tregs from a recent-onset T1D subject
|
| Data processing |
Bayesian Hierarchical Analysis (using the BGX: R package) was used to find differentially expressed genes across 12 T1D and 15 controls. Details are in the supplementary notes to the accompanying paper. For SAM analysis, we used RMA normalised intensity values as input. For analysis of 10 T1D and 10 Controls, we used adaptive MCMC methods and probe affinity effects.
|
| |
|
| Submission date |
Feb 20, 2008 |
| Last update date |
Aug 28, 2018 |
| Contact name |
Martin Hessner |
| E-mail(s) |
mhessner@mcw.edu
|
| Organization name |
Medical College of Wisconsin
|
| Department |
Pediatrics
|
| Lab |
Max McGee National Research Center for Juvenile Diabetes
|
| Street address |
8701 Watertown Plank Road
|
| City |
Milwaukee |
| State/province |
WI |
| ZIP/Postal code |
53226 |
| Country |
USA |
| |
|
| Platform ID |
GPL570 |
| Series (1) |
| GSE10586 |
Expression data from recent-onset T1D patients and controls |
|
| Relations |
| Reanalyzed by |
GSE119087 |
