The total RNA was purified with Qiagen RNeasy Mini kits (Qiagen, Hilden, Germany) according to the manufacturer's recommendations.
Label
Biotin
Label protocol
For each sample preparation, total RNA was converted into double-stranded cDNA by reverse transcription using a cDNA Synthesis System kit including an oligo(dT)24 – T7 primer (Roche Applied Science, Mannheim, Germany) and Poly-A control transcripts (Affymetrix, Santa Clara, CA, USA). The generated cRNA was purified using the GeneChip Sample Cleanup Module (Affymetrix) and quantified using the NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). The incubation steps during the cDNA synthesis, in vitro transcription reaction, and target fragmentation were performed using the Hybex Microarray Incubation System (SciGene, Sunnyvale, CA, USA) and Eppendorf ThermoStat plus instruments (Eppendorf, Hamburg, Germany).
Hybridization protocol
Hybridization, washing, and staining protocols, respectively, were performed on Affymetrix GeneChip instruments (Hybridization Oven 640, Fluidics Station FS450) as recommended by the manufacturer.
Scan protocol
Scanning was performed on Affymetrix GeneChip Scanner GCS3000 instruments as recommended by the manufacturer using default settings. The software used was GCOS 1.2 or higher.
Data processing
The Robust Multichip Average (RMA) algorithm as implemented in the R package affy (version 1.28.0) was applied to generate probe set level signal intensities. (Irizarry et al., Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics. 2003 Apr;4(2):249-64)
