RT-qPCR Analysis of Liver Tissue

Total RNA was extracted using TRIzol reagent [40 (link)]. RNA integrity was determined by electrophoresis using 1.5% agarose gels. The concentration (A260) and purity (A260/A280, A260/A230) of the RNAs were measured using a NanoDrop 1000 instrument (Thermo Fisher Scientific, Wilmington, DE, USA). The RNA was reverse transcribed into complementary DNA (cDNA) using a cDNA reverse transcription kit (Trans Gen Biotech, Shanghai, China). Each group consisted of three biological replicates, with three liver tissue samples in each replicate. RT-qPCR was conducted under standard cycle conditions, consisting of an initial denaturation step at 95 °C for 10 min, followed by 40 cycles of amplification at 95 °C for 30 s and annealing/extension at 60 °C for 30 s. The 2^−ΔΔCt method was employed for data analysis [41 (link)]. Primer sequences used in this study are shown in Table 1. β-actin is the housekeeping gene used in this study.