Total RNA was extracted from freshly purified T cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) followed by removal of genomic DNA using the RNeasy mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The purity and quality of all extracted RNA samples were confirmed by measuring A260/A280 ratio and separation on agarose gel to ensure RNA integrity prior to microarray analyses
Label
Biotin
Label protocol
First and second strand cDNA was synthesized from 5 μg high-quality, purified total RNA using a T7-(dT)24 primer and Invitrogen Life Technologies SuperScript Choice system kit (Invitrogen). Biotin-labeled cRNA was then synthesized using ENZO BioArray HighYield RNA Transcript Labeling Kit (Enzo Diagnostics Inc., Farmingdale, NY) according to the manufacturer’s instruction.
Hybridization protocol
After fragmenting, the labeled cRNA was hybridized to HG-U133 oligonucleotide array chips (Affymetrix Inc., Santa Clara, CA, USA).
Scan protocol
The arrays were then washed and stained with Streptavidin-Phycoerythrin (SAPE) (Molecular Probes Inc., Eugene, OR, USA) in an Affymetrix fluidics station. The arrays were then scanned in an Affymetrix scanner and the expression values for each probe set were estimated using the Affymetrix Microarray Suite Software (MAS v5.0).
Data processing
The signal values were imported into the GeneSpring 7.0 software tool (Silicon Genetics, Redwood City, CA, USA) to find out genes with significant differential expression. Quantile normalization with regard to 22 448 genes, PM probes and median polishing were utilized.
