Quantitative real-time RT-PCR analysis
Total RNA was extracted from liver, muscle, hypothalamus, and
periepididymal fat tissues using Trizol reagent (Invitrogen, Carlsbad,
CA, USA) and purified with RNeasy Mini Kit (Qiagen, Hilden, Germany)
following manufacturer’s protocol. cDNA was synthesized using ReverTra
Ace qPCR RT Master Mix (Toyobo, Osaka, Japan). A total of 68 genes were
chosen; if primer information was not available in the literature,
primer was designed using PRIMER3 software. GAPDH was used for
normalization. The primer sequences are provided in supplementary Table
1. qRT-PCR was performed using QuantiSpeed SYBR Green Kit (PhileKorea,
Republic of Korea).