Fast sybr green master mix system

Total RNA was extracted from cells or tissues using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. First-strand complementary DNA was synthesized using SuperScript II Reverse Transcriptase kit (Invitrogen; Thermo Fisher Scientific, Inc.). qPCR was performed using the Fast SYBR^® Green Master mix system (Roche Applied Science, Penzberg, Germany) on an ABI 7500 system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The qPCR reaction was subsequently performed according to the following conditions: Initial step, 95°C for 5 min; second step, 95°C for 10 sec, 60°C for 30 sec and 72°C for 10 sec for a total of 35 cycles. The primers used were as follows: EZH2 forward, 5′-TTTCCAACACAAGTCATCCC-3′, and reverse, 5′-ATAAACCCACATTCTCTATCCC-3′; GAPDH forward, 5′-CCGTCTAGAAAAACCTGCC-3′, and reverse, 5′-GCCAAATTCGTTGTCATACC-3′. The relative mRNA level was calculated using the 2^−∆∆Cq method and normalized to GAPDH (15 (link)). The experiment was performed in triplicate.