Gene expression levels of POMC, MeCP2 and CRH in the hypothalamic tissues were measured by quantitative real time PCR (SYBR green assay). Total RNA of each mediobasal hypothalamus (MBH) sample was extracted using RNeasy purification kit (Quiagen, Valenica CA) and converted to first strand complementary DNA (cDNA) using high capacity cDNA reverse transcription kit (Applied Biosystems, Carlsbad, CA). The primer sequences used for POMC, MeCP2, CRH and the housekeeping gene GAPDH are as described previously [21] and given in Table 1. RT- PCR was performed at 95°C for 5 min followed by 40 cycles of 95°C for 15 sec, 60°C for 30 sec, 72°C for 40 sec in Applied Biosystems 7500 Real time PCR system (ABI Carlsbad, CA). The quantity of target gene expression was measured using a standard curve. The relative expression level of each hypothalamic gene was quantitated as the ratio of GAPDH.
