Rat Genome-Wide Expression Analysis

Labeling was performed using the Agilent LowInput QuickAmp Labeling Kit One-Color (5190 − 2305; Agilent Technologies).The SurePrint G3 Rat GE 8 × 60K Kit were used (G4853A, Agilent Technologies). Briefly, first-strand cDNA synthesis was performed using an oligo(dT) 24 primer containing a T7 RNA polymerase promoter site. The cDNA was used as a template to generate Cy3-labeled cRNA that was used for hybridization. After purification and fragmentation, aliquots of each sample were hybridized to Agilent Oligo Microarrays (G2534-60014, Agilent Technologies). After hybridization, each array was sequentially washed and scanned by Agilent microarray scanner. Arrays were individually visually inspected for hybridization defects, and quality control procedures were applied as recommended by the manufacturer of the arrays. For array readout, Agilent Feature Extraction 9.5.3 Software was used, and microarray data were imported, log2-transformed and quantile normalized using robust multi-array average (RMA), and expression levels were summarized on a transcript level using average gene expression values of the replicated probes, as recently described [19 (link)]. Differential gene expression was calculated using the moderated t-test as described by Ritchie et al. [20 (link)] and implemented in the R/Bioconductor package limma.