Hifair first strand cdna synthesis supermix

The RNA was extracted using a total RNA extraction kit (LS1040, Promega, Madison, WI, United States). Following the manufacturer’s instructions, 1 µg of total RNA was reverse transcribed into cDNA using Hifair first Strand cDNA Synthesis SuperMix (11141ES10, Yeasen Biotech, Shanghai, China) for RT-qPCR analysis. Quantitative PCR was performed on a Lightcycler 96 (Roche, Basel, Switzerland) with Hieff qPCR SYBR Green Master Mix (No Rox) (11201ES03; Yeasen Biotech). The relative expression was calculated for each gene by the 2^−ΔΔCT method, normalized against GAPDH expression, and presented as fold changes relative to the control. The sequences of all the primers used in this study are shown in Supplementary Table S2.

Total RNA was extracted from tissues and cells using TRIzol (Takara, Shiga, Japan) according to the manufacturer’s instructions. RNA was reverse transcribed to cDNA using the Hifair™ First Strand cDNA Synthesis SuperMix (Yeasen, Shanghai, China). Quantitative real-time PCR was performed using the Hifair™ qPCR SYBR Green Master Mix (Yeasen) in three technical replicates. The expression values of indicated genes were normalized to GAPDH and calculated using the 2^−ΔΔCt method. The primers were listed below: GAPDH F: 5’-GGGAAGGTGAAGGTCGGAGT-3’, R: 5’-GGGGTCATTGATGGCAACA-3’; HOXA13 F: 5’-GAACGGCCAAATGTACTGCC-3’, R: 5’-GTATAAGGCACGCGCTTCTTTC-3’; ABCC4 F: 5’-GCAAAATCATCGTGTTTGTGAC-3’, R: 5’-AAAAGGTCTGGATTCTTCGGAT-3’.