Total RNA was extracted using TRIzol Reagent according to the manufacturer's 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
according to manufacturer’s manual 701880Rev4 (Affymetrix, Santa Clara, CA). Briefly, in the first cycle, double stranded cDNA was prepared with random hexamers tagged with a T7 promoter sequence followed by the generation of cRNA using the GeneChip WT Synthesis and Amplification kit (Affymetrix). cRNA concentration after cleanup was measured with the NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, DW). In the second cycle, sense oriented single-stranded DNA containing dUTP is generated and the concentration is, after cleanup, measured using the nanodrop. The cRNA is hydrolysed and the single stranded DNA is fragmented using uracil DNA glycosylase (UDG) and apurinic/apyrimidinic endonuclease 1 (APE1) (GeneChip WT terminal Labeling kit, Affymetrix). The quality of fragmentation (fragments should be between 40 and 70 nucleotides) is checked on the bioanalyzer (Agilent, Waldbronn, Germany). The fragmented DNA is labeled by terminal deoxynucleotidyl transferase (TDT) with the Affymetrix DNA Labeling reagent that is covalently linked to biotin (GeneChip WT terminal Labeling kit, Affymetrix).
Hybridization protocol
Labeled DNA was hybridized to the array during 16h at 45°C
Scan protocol
The arrays were washed and stained in a fluidics station using the GeneChip hybridization, Wash end Stain kit (Affymetrix) and scanned using the Affymetrix 3000 GeneScanner.
Data processing
All image files were generated using the Affymetrix GeneChip command console (AGCC). The raw data were analyzed with RMA sketch using the standard settings for Gene 1.0 ST arrays of Expression Console in the AGCC software (affymetrix).
