Improm rna transcriptase kit

H295R cells were cultured under normal growth and starvation conditions and total RNA was isolated using the TRIzol method according to the manufacturer’s instructions (Invitrogen Life Technologies, Carlsbad, CA, USA). RNA was reverse-transcribed to cDNA using the Improm RNA transcriptase kit (Promega) as previously described12 (link)71 (link). qRT-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). Briefly, qRT-PCR was performed in 96 well plates using 50 ng/well cDNA and 1 μl (20 pmol/μl) specific primers (Microsynth, Balgach, Switzerland) in a total volume of 25 μl. The cyclophilin A gene was used as endogenous control. Specific primer sequences may be found in Supplementary Table S1. Fold change in gene expression for a particular gene was calculated by the 2^−ΔΔCt method72 (link)73 (link). Amplification curves and the mean cycle threshold (Ct) values were calculated using the 7500 Fast System SDS software (Applied Biosystems), and correction for the endogenous gene, ΔCt and ΔΔCt were calculated as previously described12 (link).

H295R cells were cultured under GM and SM conditions, and with resveratrol and metformin treatments. Total RNA was isolated using the TRIzol method according to the manufacturer's instructions (Invitrogen Life Technologies, Carlsbad, CA, USA). cDNA was produced using the Improm RNA transcriptase kit (Promega). qRT-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), specific primers (Microsynth, Balgach, Switzerland) and 50ng mRNA in a total volume of 25μl. specific primers were newly designed using the NCBI primer designing tool Primer-Blast (https://www.ncbi.nlm.nih.gov/tools/primer-blast/). Primer sequences are given in Supplemental Material (S1 Table). Cycling conditions comprised a first incubation at 50°C for 2 min and a second incubation at 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 60 sec. GAPDH was used as endogenous control. Fold change in gene expression for a particular gene was calculated by the 2^−ΔΔCt method. Amplification curves and the mean cycle threshold (Ct) values were calculated using the 7500 Fast System SDS software (Applied Biosystems), and correction for the endogenous genes, ΔCt and ΔΔCt were calculated [19 (link)]

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)}.