Total RNA was isolated from PBMCs using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA), followed by additional purification using RNeasy from Qiagen.
Label
biotin
Label protocol
RNA is converted into cDNA using a T7 promoter-tailed oligo-dT primer in the synthesis of the first cDNA strand; second strand cDNA synthesis is then carried out. The double-stranded cDNA is used as the template in an in vitro transcription (IVT) reaction catalyzed by T7 polymerase and containing biotinylated CTP and UTP in addition to the four unmodified ribonucleoside triphosphates.
Hybridization protocol
The purified cRNA is fragmented in order to facilitate the subsequent hybridization step. The fragmented cRNA is added to a hybridization solution containing several biotinylated control oligonucleotides (for quality control), and hybridized to a microarray chip overnight at 45°C. The chips are then transferred to a fluidic station 400 (Affymetrix, Santa Clara, CA, USA) that performs washes to remove cRNA that has not hybridized to its complementary oligonucleotide probe.
Scan protocol
The DNA chips were then analyzed using a Gene Array Scanner (Affymetrix).
Description
Healthy volunteer
Data processing
CEL files were obtained using Affymetrix Microarray Suite 5.0 software; a DNA Chip Analyzer (DChip) normalized all CEL files to a baseline array with overall median intensity, and the model-based expression (perfect match only) was used to compute expression values.
