Liver Gene Expression Analysis via qRTPCR

The total RNA in each liver sample was extracted using Trizol following the manufacturer’s instructions (Accurate Biology, Changsha, China). The concentrations and purities of total RNA were determined by Spectrophotometer (Denovix DX-11, Wilmington, DE, USA). Then, the cDNA was obtained by reverse transcription kit (Accurate Biology, Changsha, China). The mRNA relative expressions of fatty-acid synthase (FASN), peroxisome proliferator activated receptor gamma (PPAR-γ), solid alcohol regulatory element binding protein 1c (SREBP1c), CCAAT/enhancer binding proteins alpha (C/EBPα), adipose triglyceride lipase (ATGL), lipoprotein lipase (LPL), hormone-sensitive lipase (HSL) and sirtuin1 (Sirt1) in the liver tissue were determined by using a LightCycler 96 fast real-time PCR system (Roch, Switzerland) and SYBR^®Green Premix Pro Taq HS qPCR Kit (Accurate Biology, Dalian, China), as previously described by Li et al. [20 (link)]. The primer sequences are presented in Table S1. The target gene’s relative mRNA expression was calculated using the threshold cycle ( $2^{-\Delta \Delta \mathrm{Ct}}$ ) method and normalized by the intrinsic reference gene (β-actin) expression.