Total RNA was extracted using the RNeasy Mini kit (Qiagen Inc., Valencia, CA, USA) and DNA was removed by on-column DNase digestion with the RNase-Free DNase set (Qiagen). RNA quality and concentration was determined by analysis with an Agilent 2100 bioanalyzer.
Label
Cy3
Label protocol
cDNA synthesis followed by Cy3-labeling
Hybridization protocol
Cy3-labeled cDNA was hybridized overnight at 42°C to mus musculus 12x135K Array (Roche NimbleGen)
Scan protocol
Arrays were scanned with the Axon GenePix 4000B microarray scanner
Data processing
The raw data (.pair file) was subjected to RMA (Robust Multi-Array Analysis; Irizarry et al. Biostatistics 4(2):249), quantile normalization (Bolstad et al. Bioinformatics 19(2):185), and background correction as implemented in the NimbleScan software package, version 2.5 (Roche NimbleGen, Inc.).
