Quantitative Expression Profiling of mRNA and miRNA

Quantitative RT-PCR (Q-PCR) was used to measure mRNA and microRNA (miRNA) expression levels (primers shown in Supplementary Table S1). Total RNA was extracted from adipose tissues using TRIzol reagent (Invitrogen) and further purified with RNeasy columns (Qiagen) according to the manufacturer’s protocol. Reverse transcription of mRNA and miRNA was performed using the PrimeScript RT Master Mix kit and the PrimeScript^™ miRNA RT-PCR Kit, respectively (both obtained from TaKaRa, Dalian, China) following the manufacturer’s recommendations. Quantitative PCR was performed using the SYBR Green Real-time PCR Master Mix (TaKaRa) on a CF96 Real-Time PCR Detection System (Bio-Rad, Hercules, California, USA). The ACTB, TBP and TOP2B genes were simultaneously used as internal controls for mRNA normalization. Expression levels of U6 served as endogenous controls for miRNA expression and were utilized to normalize the corresponding data. The 2^−ΔΔCt method was used to determine the relative mRNA and miRNA abundance30 (link).