Ligthcycler 96 instrument

Total RNA from HepG2 cells, liver, and epididymal adipose tissue was extracted with Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. RNA quantity and quality were measured with a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Total RNA (2 μg) was reverse-transcribed to complementary DNA (cDNA) in a 50-μL final volume using 690-ng random primers, 0.72 mM deoxynucleotide triphosphate (dNTP) mix, 1× first-strand buffer, 3.6 mM dithiothreitol (DTT), 5 U of RNAase inhibitor, and 260 U Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase (Invitrogen, Carlsbad, CA, USA). The reaction was incubated for 10 min at 25 °C, 60 min at 37 °C, 15 min at 70 °C, and 5 min at 4 °C. Afterward, cDNA was used as a template for amplification in real-time PCR reactions. Amplification was carried out in a 10-μL final volume containing 2 μL of cDNA, 1× Universal PCR Master Mix, and 1× specific Taqman primer/probe (Applied Biosystems, Foster City, CA, USA) for enlisted genes in Table S1 according to the manufacturer’s directions using a LigthCycler 96 instrument (Roche Molecular Systems, Pleasanton, CA, USA). The messenger RNA (mRNA) expression mean of duplicates per sample was normalized to 18S. Data were analyzed using the 2-Δct method [34 (link)].