Quantitative RT-PCR Analysis of Gene Expression

Total RNA was extracted from cells using RNAiso Plus (Takara) and treated with DNase I (RNase-free) (Takara) according to manufacturer's instructions. 0.5 μg total RNA was reverse transcribed to cDNA using Reverse Transcriptase Kit (M-MLV) (ZOMANBIO). The cDNA was diluted 1:10 prior to PCR amplification and then subjected to real time PCR in a Bio-Rad CFX96 Real-Time PCR Detection System using SYBR Green PCR Master Mix (Bio-Rad) as described previously (24 (link)). The primers used for qRT-PCR were listed in Supplementary Table S2. The relative mRNA levels were determined by the ΔΔCt quantification method using the CFX manager 3.1 (Bio-Rad). Actin mRNA levels were used as internal controls. The validity of the qRT-qPCR data was assured by following the MIQE guidelines (25 (link)).