Accupower greenstar rt qpcr master mix

To examine the effect of TNSO on adipogenic gene expression, 3T3-L1 preadipocytes were allowed to differentiate for 5 days with 0.75 μL/mL TNSO. TNSO was added on days 0, 2, and 4, whereas the control group was treated only with DMSO. On day 5, post-differentiation induction, total RNA of 3T3-L1 cells was extracted using the RNeasy Mini Kit (QIAGEN, Hilden, Germany). Target RNA was amplified using the AccuPower® GreenStar™ RT-qPCR Master Mix (Bioneer, Daejeon, Korea). Quantitative RT-PCR was performed on LightCycler® 96 system (Roche, Basel, Switzerland) as follows: reverse transcription at 60 °C for 15 min, pre-denaturation at 95 °C for 5 min, denaturation at 95 °C for 10 s, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s. The denaturation step to extension step was cycled 45 times. Relative mRNA expression levels were normalized to those of β-actin as the internal control. The sequences of the primers were as follows: 5′-GAGGTATCCTGACCCTGA AGTA-3′ (β-actin forward), 5′-CACACGCAGCT CATTGTAGA-3′ (β-actin reverse), 5′-TGGAAGACAGCTCCTCCTCG-3′ (FABP4 forward), 5′-AATCCCCATTTACG CTGATGATC-3′ (FABP4 reverse), 5′-ACCTGGTAGACCACTGCATTGAC-3′ (FAS forward), 5′-CCTGATGAAACGACACATTCTCA-3′ (FAS reverse), 5′-TGAGACAG GAGATGTTGGAATG-3′ (adiponectin forward), 5′-ACGCTGAGCGATACACATA AG-3′ (adiponectin reverse).