Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

Total RNA was extracted from the cells subjected to different treatments, using Trizol (Takara, Tokyo, Japan). The mRNA was reverse-transcribed to complementary DNA (cDNA) using the PrimeScript RT-PCR kit (Takara) according to the manufacturer’s s instructions. The qRT-PCR analysis was performed with SYBR Premix Ex Taq II (Tli RNaseH Plus; Takara) in a Light Cycle Roche 480 II Real-time PCR system (Roche, Basel Switzerland) in triplicate. The primer sequences used for qRT-PCR analysis are listed in Table 1. The expression levels of target genes were normalized to those of GAPDH. The relative expression level was calculated using the 2^–ΔΔCt method.

Primer sequences for qRT-PCR.