RNA Extraction and qPCR Analysis

Total RNA was extracted using an RNeasy Plus Micro Kit (QIAGEN, 74034) according to the manufacturer’s instructions^{15 (link)}. Cell lysates were spun using the kit’s gDNA Eliminator spin columns to remove genomic DNA. Total RNA was purified using RNeasy MinElute spin columns. First-strand cDNA was then synthesized from 1 µg of RNA using PrimeScript RT Master Mix (Takara, RR036A). Then 20 µl reactions were prepared by combining 4 µl of PrimeScript RT Master reaction mix, 2 µl of gene-specific enhancer solution, 1 µl of reverse transcriptase, 1 µl of gene-specific assay pool (20 ×, 2 µM), and 12 µl of RNA diluted in RNase-, DNase-, and genomic DNA-free water. We performed qPCR using TB Green Premix Ex Taq II (Takara, RR820Q) on the C1000 Touch Thermocycler CFX96 Real-Time System (Bio-Rad). Analysis was performed using Bio-Rad CFX Manager software 2.0. The data were normalized to RNA GAPDH and the fold change was calculated via the 2^−DDCt method. The relative concentrations of mRNA were expressed in arbitrary units based on the untreated group, which was assigned a value of 1.