A balanced dye-swap design was adopted for our microarray experiment. For each brain region, every RNA sample from the control group was randomly paired with a sample from the treatment group. Then, each of the paired samples was split into two aliquots, which were labeled separately with either Cy3 or Cy5. Next, the Cy3-labeled control probes and Cy5-labeled treatment probes were combined and hybridized to the same slide; the Cy5-labeled control probes and Cy3-labeled treatment probes were combined and hybridized to another slide. The probe was labeled and hybridized with the cDNA microarray using a 3DNA Array 900™ Expression Array Detection kit (Genisphere, Hatfield, PA) according to the manufacturer’s protocol. Hybridized slides were first washed in 1× SSC–0.1% SDS buffer at 60°C for 10 min and then in 2× SSC–0.2× SSC buffer for 10 min at room temperature prior to being spun down briefly and scanned on a GenePix 4000B scanner (Axon Instruments Inc., Union City, CA). A total of 60 arrays were processed for this study with 10 slides per brain region.
