Mj real time pcr system

Total RNA was isolated from the cells using RNAiso Plus (Takara Bio, Inc., Otsu, Japan), according to the manufacturer's protocol. Total RNA (5 µg) was reverse transcribed into cDNA using the M-MLV First-Strand Synthesis system (Promega Corporation, Madison, WI, USA). cDNA was analyzed in triplicate using the MJ Real-Time PCR system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The primers used are shown in Table I. qPCR was performed using the Power SYBR Green Master Mix (Takara Bio, Inc.) and the ABI 7300 Real-Time PCR detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.). PCR conditions were as follows: Initial denaturation step at 95°C for 15 sec, followed by 40 cycles of amplification and quantification at 95°C for 10 sec, 60°C for 30 sec and 72°C for 30 sec. Fold changes in expression of each gene were calculated using the comparative quantification method (19 (link)).

RT-qPCR analysis was performed using standard procedures. Total RNA was isolated using a RNeasy kit (Qiagen, Inc.), 5 µg of each sample was reverse-transcribed using the M-MLV first-stand synthesis system (Invitrogen; Thermo Fisher Scientific, Inc.) and cDNAs were analyzed in triplicate with the MJ Real-Time PCR System (Bio-Rad Laboratories, Inc.). The sequences of the gene-specific and GAPDH primers used for qPCR are presented in Table I. Amplification was performed for 40 cycles with a denaturation temperature of 94°C for 30 sec, annealing temperature of 58°C for 30 sec and extension temperature of 74°C for 45 sec in a Veriti thermal cycler (Applied Biosystems; Thermo Fisher Scientific, Inc.). The PCR products were 200 bp in length. RT-qPCR was performed using the Power SYBR Green Master Mix (Takara Bio, Inc.) and an ABI 7300 Real-Time PCR system (Applied Biosystems, Thermo Fisher Scientific, Inc.). All primers were synthesized by Invitrogen (Thermo Fisher Scientific, Inc.). Fold changes in expression of each gene were calculated by a comparative threshold cycle (Cq) method using the formula 2^−ΔΔCq (28 (link)). Data were collected from three independent experiments.