Sybr green rox qpcr master mix

The relative expression level of STCAT16 was determined by RT-qPCR. Total RNA was extracted from 100 mg tissues or cells using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.). Subsequently, RNA was reverse-transcribed into cDNA with a Moloney murine leukaemia virus reverse transcriptase kit (Thermo Fisher Scientific, Inc.). The temperature protocol was as follows: 42°C for 60 min, 70°C for 5 min. According to the manufacturer's instructions, qPCR was executed using SYBR-Green I (Takara Biotechnology, Co., Ltd., Dalian, China) and an Applied Biosystems 7500 Real-Time PCR System (Thermo Fisher Scientific, Inc.). The qPCR reaction mixture contained 10 µl SYBR-Green/ROX qPCR Master Mix (Takara Biotechnology, Co., Ltd.), 1 µl forward primers (STCAT16, 5′-CATCAAGGCTTGTGGGATGT-3′; GAPDH, 5′-CTGGGCTACACTGAGCACC-3′), 1 µl reverse primers (STCAT16, 5′-AAGCCGAAAGGTCAACTGC-3′; GAPDH, 5′-AAGTGGTCGTTGAGGGCAATG-3′), 2 µl cDNA and 6 µl sterile double steamed water. GAPDH was used as a reference gene. The thermocycling conditions were the following: 95°C for 10 min, followed by 40 cycles at 95°C for 15 sec and 60°C for 60 sec. Primers of STCAT16 were synthesized by Shanghai Ruian BioTechnologies Co., Ltd. (Shanghai, China). The data were quantified using the 2^−ΔΔCq method (24 (link)). Each experiment was performed in triplicate.