As previously described (Chang et al., 2008), total RNA from pooled microdissected hypothalamic samples was extracted with TRIzol reagent. RNA was treated with RNase-free DNase I before RT, and cDNA and minus RT were synthesized using an oligo-dT primer with or without SuperScript II reverse transcriptase. The real-time PCR was performed with Applied Biosystems’ system. With Applied Biosystems Primer Express V1.5a software, primers were designed to have a melting temperature of 58-60°C and to produce an amplicon of 50-150 bp. The last five bases on the 3’ end contained no more than 2 G and/or C bases to reduce the possibility of nonspecific product formation. For orexin (OX), the primer pairs (5’-AGATACCATCTCTCCGGATTGC-3’ and 5’ CCAGGGAACCTTTGTAGAAGGA-3’) (GenBank #AF019565) generate a 73 bp amplicon corresponding to the nucleotide 48-121 of the sequence. For the house-keeping genes, the primer pairs for β-actin (5’-GGC CAA CCG TGA AAA GAT GA-3’ and 5’-CAC AGC CTG GAT GGC TAC GT-3’ (GenBank #NM031144) generate a 79 amplicon corresponding to the nucleotide 420-498 of the sequence that crosses exon 2 and exon 3. The SYBR Green PCR core
