Double-stranded cDNA was synthesized

from RNA isolated using the MessageAmpTM aRNA Kit (Ambion, Austin, TX). Biotinylated cRNA was in vitro transcribed from double-stranded cDNA template check details using MegaScript High-Yield Transcription Kit (Ambion). Resulting cRNA (15 μg) was purified using the MessageAmpTM aRNA Kit and fragmented before hybridization to Affymetrix GeneChip MGU74Av2 microarrays (12,488 probes). GeneChips were washed and stained with streptavidin phycoerythrin according to manufacturer’s instructions prior to scanning with an Agilent Gene Array scanner. Microarray data analysis Quality control analysis of microarray gene expression data was performed as recommended by Bolstad et al. [66]. Briefly, microarray data quality was assessed using the following plots: box, histogram, MA, RNA degradation, housekeeping gene, Relative Log Expression (RLE) and Normalized Unscaled Standard Error (NUSE). selleck compound None of the microarrays were found to be significant outliers and unsupervised clustering of microarrays revealed no significant batch effects. In addition,

physical chip images revealed no manufacturing or spatial artifacts. In short, all microarrays passed quality control checks and were retained for further analysis. Microarray gene expression data was deposited at the Gene Expression Omnibus (http://​www.​ncbi.​nlm.​nih.​gov/​geo/​) at the National Center for Biotechnology Information with accession number GSE40379. Microarray data was transformed to the log2 scale and normalized using the GC Robust Multichip Average (GCRMA) method [67]. Fold changes were initially calculated by dividing expression levels in DBA/2 mice by those in C57BL/6 mice at each time point (day 0, 10, 14, and 16). A positive ratio indicates greater expression in DBA/2 mice compared to C57BL/6

mice but does not necessarily equate to upregulation. For example, a gene might be constitutively Isoconazole expressed prior to infection (day 0) in both strains and then following infection downregulated less in DBA/2 mice compared to C57BL/6 mice. This would result in a positive ratio indicative of higher expression in DBA/2 than C57BL/6 even though the gene is downregulated compared to the uninfected control (day 0). Therefore, fold changes were also calculated by dividing post-infection time points (day 10, 14 and 16) by the uninfected control (day 0) in order to confirm the direction of gene expression changes. In addition, abnormally high fold change values may result when expression levels below the limit of detection are used as the denominator in fold change calculations. The limit of detection for this study was calculated as an expression level of 35.3, which was the 95th percentile expression level of the absent and marginal probes identified using the MAS 5 algorithm from Affymetrix [68].