Porcine v2 gene expression microarrays

Total RNA was reverse transcribed after priming with a DNA oligonucleotide containing the T7 RNA polymerase promoter 5’ to a d(T)24 sequence. After second-strand cDNA synthesis and purification of double-stranded cDNA, in vitro transcription was performed using T7 RNA polymerase. The quantity and quality of the cRNA was assayed by spectrophotometry on the Agilent Bioanalyzer as indicated for Total RNA analysis. cRNA was fragmented to uniform size and hybridized to Porcine (V2) gene expression microarrays (Design ID-026440, Agilent Technologies). Slides were washed and scanned on an Agilent SureScan Microarray Scanner. Expression values were extracted using Agilent Feature Extraction software and data were analyzed with GeneSpring GX software (Agilent Technologies). Array normalization and batch correction were performed using COMBAT-Quantile R-script in GeneSpring. Data analysis was also performed in GeneSpring. Time-point data from the same animals were analyzed using repeated measures ANOVA. Multiple testing correction was performed using Benjamini Hochberg False Discovery Rate (FDR). Principal component analysis (PCA) plots were generated reducing the data to three principal components.