|
| Status |
Public on Jan 10, 2010 |
| Title |
33 preN |
| Sample type |
RNA |
| |
|
| Source name |
Peripheral blood mononuclear cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Peripheral blood mononuclear cells
disease state: healthy
|
| Extracted molecule |
total RNA |
| Extraction protocol |
PBMCs were isolated from edetic anticoagulated whole blood by centrifugation over LymphoprepTM separation medium (Axis-Shield, Oslo, Norway) and were subjected to RNA extraction using the TriReagent® as instructed by the manufacturer (Ambion, Austin, TX). The time delay from a blood draw to RNA extraction of PBMCs was about 30 minutes.
|
| Label |
biotin
|
| Label protocol |
Double strand cDNA was synthesized from 1 ug of total RNA using a cDNA Synthesis System (InvitroGen, Basel, Switzerland) with the T7-(T)24 primer. The in vitro-labeling kit (Enzo; Farmingdale, NY) was used to transcribe the cDNA into cRNA in the presence of Biotin-11-CTP and Biotin-16-UTP according to the instructions supplied with the kit.
|
| |
|
| Hybridization protocol |
Twelve ug fragmented cRNA was then used for hybridization. Hybridization and staining were performed as suggested by manufaturer.
|
| Scan protocol |
Hybridized Arrays were scanned using an Affymetrix GeneChip 3000 scanner.
|
| Description |
We conducted a genome-wide transcription analysis in peripheral blood mononuclear cells (PBMCs) from 14 women (9 MS patients and 5 normal controls) followed during their pregnancy. Samples were obtained before pregnancy and at the third, sixth, and ninth month of gestation. Findings were corroborated by real time RT-PCR in a larger cohort of MS patients (n=28) and healthy controls (n=11). Afterwards, we compared expression profiles of patients relapsing during pregnancy (n=23) with those of relapse-free patients (n=5).
|
| Data processing |
GeneChip Operating Software (GCOS) (Affymetrix, Santa Clara, CA) was used to generate background-normalized image data (CEL files). Microarray quality controls and statistical validation was done using Bioconductor. The presence of hybridization/construction artifacts was evaluated with the fitPLM function (Bioconductor package affyPLM), and the probe (PM) intensity distribution was evaluated using hist function (Bioconductor package affy). Probe set intensities were obtained by means of RMA (Robust Multichip Average) and normalization was done according to the quantiles method.
|
| |
|
| Submission date |
Jul 29, 2009 |
| Last update date |
Jan 10, 2010 |
| Contact name |
Raffaele A Calogero |
| E-mail(s) |
raffaele.calogero@unito.it
|
| Phone |
++39 0116706454
|
| Organization name |
University of Torino
|
| Department |
Molecular Biotechnology Center
|
| Lab |
Bioinformatics and Genomics Unit
|
| Street address |
Via Nizza 52
|
| City |
Torino |
| State/province |
To |
| ZIP/Postal code |
10126 |
| Country |
Italy |
| |
|
| Platform ID |
GPL571 |
| Series (3) |
| GSE17393 |
Transcription signature of Multiple Sclerosis in peripheral blood mononuclear cells. |
| GSE17409 |
Pregnancy changes expression in peripheral blood mononuclear cells of healthy donors |
| GSE17449 |
Transcription signature of Multiple Sclerosis |
|
