whole blood from venipuncture from an healthy subject after informed consent
Treatment protocol
Whole blood was collected using a PAXgene collection tube (PreAnalytix/Qiagen, Courtaboeuf, France)
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted with PAXgene Blood RNA kit (Qiagen, Courtaboeuf, France), including DNase I treatment. Globin RNA was removed using the GLOBINclear kit (Ambion, Austin, TX, USA). Two micrograms of total RNA, cleared from globin RNA, were used.
Label
biotin
Label protocol
Ribosomal RNA reduction, first and double strand cDNA synthesis, cRNA synthesis, second round single-strand (ss) cDNA synthesis, ss-cDNA fragmentation and labelling were processed following the manufacturer’s instructions (http://www.affymetrix.com/products/arrays/specific/exon.affx).
Probe arrays were scanned using the Affymetrix GCOS 3000 7G and the Gene-Chip Operating Software v.1.4 to obtain the .CEL files
Description
exon level expression; analysis conducted with Affymetrix probe group file HuEx-1_0-st-v2.r2.pgf
Data processing
Expression signal values and p-values were obtained from .cel files for each probeset using the Robust Microarray Analysis (RMA) algorithm in ArrayAssit software (Stratagene). Normalisation of the 4 whole blood samples data of the serie was done together with 11 triplicates of human normal tissues: breast, cerebellum, heart, kidney, liver, muscle, pancreas, prostate, spleen, testes, thyroid (33 samples) (data from Affymetrix, http://www.affymetrix.com/support/technical/sample_data/exon_array_data.affx). Background was discarded with Detection Above Background (DABG) filter in Array Assist on the following criteria: 3 arrays with a p-value less or equal than 0.05.
