The approach used here to identify ethanol-responsive genes was somewhat unorthodox for a microarray study. Rather than comparing two treatment groups composed of multiple biological replicates, our treatment groups comprised relatively large samples of 29–36 genetically unique individual strains. Although only single arrays were performed per strain/treatment, the issue of biological variability was reduced by pooling tissue samples from at least 4 biological replicates per strain. Our use of the S-score analysis method to compare ethanol vs. saline responses further improved the robustness of our genetic correlation analysis [70]. Gene expression correlations or expression-genotype correlation significance were also empowered by the number of strains compared. The approach identified a robust set of genes whose expression levels were significantly altered by ethanol across the BXD strains in at least one of the three profiled brain regions. This gene set included a large contingent of our prior 2-group microarray study of brain ethanol-responsive genes [21]. Strikingly, nearly a quarter of the ethanol-responsive genes defined here were previously identified as having basal expression differences in a meta-analysis by Mulligan et al. [71] of
