Separate preparations of total RNA were made from the ACB of individual animals. Samples were not pooled. Standard Affymetrix protocols (GeneChip® Expression Analysis Technical Manual, Rev. 5 and updates) were used to synthesize biotinylated cRNA, starting with 5 μg total RNA from each region, using the Affymetrix kits for cDNA synthesis, in vitro transcription and sample cleanup. Fifteen μg of fragmented, biotinylated cRNA from each independent sample were mixed into 300 μl of hybridization cocktail, of which 200 μl were used for each hybridization. Hybridization was for 17 hr at 42°C. Samples were hybridized to the Affymetrix GeneChip® (Rat Genome 230 2.0 array GeneChips). Washing and scanning of the GeneChips were carried out according to standard protocols, as previously described (Edenberg et al., 2005; McClintick et al., 2003). To minimize potential systematic errors, all stages of the experiment were balanced across experimental groups. That is, equal numbers of animals in each group were sacrificed within the same 2-hr time frame each day, and equal numbers of RNA preparations from the representative groups were processed through the labeling, hybridization, washing and scanning protocols on a given day, in a counterbalanced order, using premixes of reagents.
