Quantitative RT-PCR Analysis of Gene Expression

Total RNA was extracted and purified from cultured cells or tissues using the RNeasy Plus Mini Kit (QIAGEN, 74136) according to the manufacturer’s instructions. The RNA was quantified by determining absorbance at 260 nm. One microgram of total RNA from each sample was reverse-transcribed into cDNA using the iScript cDNA synthesis kit (Bio-Rad, 170-8891) in a volume of 20 μl; cDNA from cell samples was amplified. The qPCR was performed using SsoFast EvaGreen Supermix (Bio-Rad, 172-5204) on the C1000 Touch Thermocycler CFX96 Real-Time System (Bio-Rad) according to the manufacturer’s protocol. Analysis was performed using Bio-Rad CFX Manager software 3.1 (Bio-Rad). The specific primers were listed in Supplementary Table 1. The gene expression was calculated via the 2^−ΔΔCt method and normalized to 18SRNA/18srna^{72 (link)}. The relative concentrations of mRNA were expressed in arbitrary units based on the untreated group, which was assigned a value of 1.