Quantifying Gene Expression in H295R Cells

Total RNA was extracted from adherent H295R cells cultured under normal growth, serum starvation and metformin treatment conditions using the TRIzol reagent according to the manufacturer’s instructions (Invitrogen Life Technologies, Carlsbad, CA, USA), followed by reverse transcription using the Improm RNA transcriptase kit (Promega, Madison, WI, USA) as previously described^{23 (link), 26 (link)}. Quantitative RT-PCR analysis was performed on the 7500-Fast real-time PCR System (Applied Biosystems, Foster City, CA, USA) using ABsolute SYBR Green Mix (ABgene; Thermo Fisher scientific, Waltham, MA, USA). As endogenous control, the GAPDH gene was used. Fold change in gene expression was calculated by the 2^−ΔΔCt method^{69 (link)}.