The total RNA was purified either with Qiagen RNeasy Mini kits (Qiagen, Hilden, Germany) or with TRIzol-based protocols
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
Data pre-processing included a summarization and quantile normalization step to generate probe set level signal intensities for each microarray experiment and was performed as previously published by Liu WM et al. PQN and DQN: algorithms for expression microarrays. J.Theor.Biol. 2006;243:273-278.
The signal used was DS, see Liu, W.-m., R. Li, J. Z. Sun, J. Wang, J. Tsai, W. Wen, A. Kohlmann, P. M. Williams, PQN and DQN: Algorithms for expression microarrays, J. Theoretical Biol., 243 (2006), 273-278.
ABS_CALL
DETECTION P-VALUE
VALUE
The signal used was DQN3, i.e., DQN signal normalized with quantiles of the beta distribution with parameters p=1.2 and q=3, see Liu, W.-m., R. Li, J. Z. Sun, J. Wang, J. Tsai, W. Wen, A. Kohlmann, P. M. Williams, PQN and DQN: Algorithms for expression microarrays, J. Theoretical Biol., 243 (2006), 273-278.
