Primescript rt reagent kit

Total RNA from cells was extracted using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.). The generation of cDNA was performed using PrimeScript RT reagent kit (TransGen Biotech, Co., Ltd.) according to the manufacturer's instructions. The qPCR was performed using SYBR^® Premix Ex Taq (Takara Bio, Inc.) with GAPDH as an endogenous control. The thermocycling conditions were as follows: 95˚C for 3 min followed by 45 cycles at 95˚C for 7 sec, 57˚C for 10 sec, 72˚C for 15 sec. Relative quantification was calculated by the ΔΔCT method (28 (link)). The following primers were used for RT-qPCR: SETDB-1 forward, 5'-taagacttggcacaaaggcac-3' and reverse, 5'-tccccgacagtagactctttc-3' and GAPDH forward, 5'-ggagcgagatccctccaaaat-3' and reverse, 5'-ggctgttgtcatacttctcatgg-3'.

Total RNA was extracted from tissues or cells using TRIzol total RNA isolation reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Subsequently, the PrimeScript RT reagent kit (Beijing Transgen Biotech Co., Ltd.) was used for RT of the RNA into cDNA, according to the manufacturer's protocol. SYBR-Green RT-qPCR Master Mix was used as the flurophore. RT-qPCR was performed using specific primers for miR-101 (forward, 5′-GTACAGTACTGTGATAACTGA-3′ and reverse, 5′-TGCGTGTCGTGGAGTC-3′), mTOR (forward, 5′-TCGGTGCAAACCTACAGAAGC-3′ and reverse, 5′-TGCAGGTCGTATATGGACAGAG-3′) and GAPDH (forward, 5′-GGAGCGAGATCCCTCCAAAAT-3′ and reverse, 5′-GGCTGTTGTCATACTTCTCATGG-3′) used as the internal control. The thermocycling conditions were as follows: Pre-denaturation at 94°C for 30 sec, followed by 45 cycles of denaturation at 94°C for 5 sec, annealing at 60°C for 15 sec and extension at 72°C for 10 sec. Each sample was tested in quadruplicates. The relative expression of each gene was quantified using the comparative quantification cycle method as follows: Copy number of target gene = 2^−ΔΔCq, ΔCq = C_{qtarget gene}−Cq_{reference gene}, ΔΔCq = ΔCq_{experimental group}−ΔCq_{control group} (23 (link)).