CD4+ T cells were isolated from blood samples by using RosetteSep CD4+ enrichment kit (StemCell Technologies, Meylan, France); purity: > 90% as determined by flow cytometry. RNA was isolated with Trizol(r) method according to the manufacturer's protocol. The RNA was purified with the RNeasy MiniElute Cleanup Kit (Qiagen, Hilden, Germany)
Label
biotin
Label protocol
In all, 5ug of total RNA was used to generate doublestranded cDNA with a T7-(dT)24-oligonucleotide primer (Sigma-ARK, Steinheim, Germany) performed with the Superscriptt double stranded cDNA-synthese Kit (Invitrogen Life Technologies). After purification with the Sample Cleanup Module (Affymetrix, Santa Clara, USA), cDNA served as a template to prepare biotinylated cRNA via in vitro transcription, using the BioArrayt HighYieldt RNA Transcript Labeling Kit (Enzo Biochem, Farmingdale, USA). The labeled cRNA transcripts were purified using the Sample Cleanup Module (Affymetrix).
Hybridization protocol
Fragmentation of cRNA transcripts and hybridization of the high-density oligonucleotide microarrays (HG-U133A arrays, Affymetrix) were performed according to the manufacturer's GeneChips Expression Analysis Technical Manual (Affymetrix).
Scan protocol
Scanning of the high-density oligonucleotide microarrays (HG-U133A arrays, Affymetrix) were performed according to the manufacturer's GeneChips Expression Analysis Technical Manual (Affymetrix).
