Hepatic Gene Expression Analysis

Hepatic samples were homogenized in TRIzol reagent and total RNA was extracted using Invitrogen PureLink RNA Mini Kit (ThermoFisher, Waltham, MA, USA). RNA concentration and purity were determined by a Biodrop Touch Duo spectrophotometer (Montreal Biotech Inc., Dorval, QC, Canada) [31 (link)]. Complementary DNA was obtained by reverse transcripting 1 μg of RNA with the Superscript VILO Master Mix (Invitrogen, Waltham, MA, USA). Gene expression was analyzed by quantitative RT-PCR using the 7500 Fast Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). The thermal profile included an initial denaturation at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 3s, and annealing and extension at 60 °C for 30 s. Amplified gene expressions were quantified by fluorescence using the PowerUp SYBR Green Master Mix (Life Technologies, Carlsbad, CA, USA) [31 (link)]. Levels of expression of target-gene mRNAs were calculated by the 2^−∆∆CT method. The list of all primers can be found in the Supplementary Materials (Table S1).