Primescript rt system kit for real time pcr

Total RNA was isolated from cultured cells using the TRIzol reagent (Invitrogen, Shanghai, China) according to the manufacturer’s protocol. The RNA was reverse-transcribed into cDNA using the PrimeScript RT System Kit for Real-Time PCR (TaKaRa, Dalian, China). The mRNA levels were measured by qRT-PCR using SYBR^® Premix Ex Taq™ (TaKaRa, Dalian, China). Relative expression levels were calculated by the 2^−ΔΔCt method with β-actin as the endogenous control. The primers are listed in Table 1.

TRIzol (Invitrogen) was used to isolate total RNA from the three stages of cultured GCs at 0 h and in two methods at 48 and 96 h. The RNA was reverse-transcribed using a PrimeScript™ RT system kit for real-time PCR (TaKaRa, Japan) according to the manufacturer’s instructions. qPCRs were performed in a CFX96TM Real-Time system (Bio-Rad, CA, U.S.A.) using a SYBR PrimeScript™ real-time PCR kit (TaKaRa). qPCRs were performed in a 25 μl reaction volume that included 2.0 μl of cDNA, 12.5 μl of SYBR Premix EX Taq, 8.5 μl of sterile water, and 1.0 μl of each gene-specific primer. The raw results were repeated three times and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using the 2^−△△Ct method [28 (link)]. Primers for the tested genes are listed in Table 1.