Hepatic FA Metabolism-Related Gene mRNA Levels

Total RNA of the liver was extracted with the TRI Reagent® (Sigma-Aldrich, St. Louis, MO, USA), according to instructions from the manufacturer. The quality and quantity of total RNA were evaluated by measuring the OD 260/280 ratio on a BioTek epoch reader with the Gen5TM Take3 Module (BioTek Instruments, Winooski, VT, USA). The concentration of total RNA was adjusted to 4000 ng/µL and then reverse-transcribed with a RevertAid First Strand cDNA Synthesis kit (#K1621, ThermoFisher Scientific, Waltham, MA, USA). The concentration of complementary (c)DNA was calculated by the BioTek epoch reader with the Gen5TM Take3 Module system and adjusted to 50 ng/µL. The resulting cDNA was amplified in a 96-well polymerase chain reaction (PCR) plate using SYBR Green/ROX qPCR Master Mix (2X) (ThermoFisher Scientific) on a QuantStudio 1 Real-Time PCR System (ThermoFisher Scientific). Gene levels were normalized to βactin, and the ratio to β-actin was calculated by setting the value of the NC group to 1. Information on primers is given in Table 10.
