Total RNA was extracted using TRIzol Reagent according to the manufacturerÆs protocol (Gibco BRL, Carlsbad, CA), followed by a cleanup procedure with RNeasy columns (Qiagen, Cologne, Germany).The total RNA quantity and quality was determined using the NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, DW) and the 2100 Bioanalyzer (Agilent, Waldbronn, Germany), respectively. Total RNA profiles of all tested samples were similar with sharp 18S and 28S rRNA peaks on a flat baseline.
Label
Biotin
Label protocol
Cellular mRNA was reverse transcribed into cDNA (SuperScript Choice System Invitrogen, Carlsbad, CA using oligo-dT primers and a T7 RNA polymerase promoter site). Two ??g of total RNA was used to prepare biotinylated cRNA with IVT labeling kit (Affymetrix, Santa Clara, CA) according to the Genechip expression analysis technical manual 701025 Rev.5. The concentration of labelled cRNA was measured using the NanoDrop ND-1000 spectrophotometer. Labeled cRNA was fragmented in a fragmentation buffer during 35 min at 94??C. The quality of labelled and fragmented cRNA was analysed using the Agilent bioanalyzer 2100.
Hybridization protocol
Fragmented cRNA was hybridised to mouse 430 2.0 arrays (Affymetrix) during 16h at 45??C.
Scan protocol
The arrays were washed and stained in a fluidics station (Affymetrix) and scanned using the Affymetrix 3000 GeneScanner.
Description
Mouse thymus
Data processing
All image files were analysed using the affy library of the BioConductor project (no background adjustment, probe-level quantile normalization, and average difference summarization)
