Separate preparations of total RNA were made from individual CNS regions from each animal. Samples were not pooled. Standard Affymetrix protocols (GeneChip® Expression Analysis Technical Manual, Rev. 5 and updates) were used to synthesize biotinylated cRNA, using the Affymetrix kits for cDNA synthesis, in vitro transcription and sample cleanup. The fragmented, biotinylated cRNA from each independent sample was mixed into 300 μl of hybridization cocktail, of which 200 μl were used for each sample. Hybridization was carried out for 17 hr at 42°C. Samples were hybridized to the Affymetrix Rat Genome 230 2.0 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).
