POMC methylation was performed by SYBR green real time PCR method of methylation specific PCR (MSP) as it was described previously [21]. DNA was extracted from MBH samples using DNeasy purification kit (Quiagen, Valenica CA). The bisulfite conversion of DNA was performed using EZ DNA methylation kit (Zymo Research, Orange, CA). Primer sequences specific for methylated and unmethylated DNA were used as described previously [21] and listed in Table 1. Rat highly methylated, low methylated control DNA (Epigen Dx, Worcester MA) also underwent bisulfite conversion and were used for preparing a standard curve. Real time quantitative 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. Relative quantity of methylated and unmethylated DNA were measured using a standard curve. DNA methylation was measured as a ratio of methylated versus unmethylated DNA.
