Quantitative Gene Expression Analysis

RNA was isolated and extracted using the RNA extraction reagent (QIAGEN, Germany) according to the manufacturer’s protocol. The genomic DNA (gDNA) was cleaved using the RNAse-Free DNase Set from QIAGEN, a company based in Germany. The RNA samples were assessed for purity using the NanoDrop 2000c (Thermo Fisher Scientific, United States) and gel electrophoresis. The reverse transcription PCR was performed using the PrimeScriptTM II 1st Strand cDNA Synthesis Kit from TaKaRa, a company based in China. The SYBR Premix Ex TaqTM II, manufactured by TaKaRa in China, was employed for quantitative reverse transcription polymerase chain reaction (qRT-PCR) on a 7,500 series real-time fluorescence quantitative cycler manufactured by Bio-RAD in the United States of America. The primers for the qRT-PCR test were prepared using the Primer Premier 5.0 software. The ACTIN gene served as the reference gene (Song et al., 2021 (link)). The primers utilized for qRT-PCR analysis are listed in Supplementary Table S2. Each experiment was repeated three times in triplicate, and a total of three biological replicates were undertaken. The gene expression levels were determined using the 2^-△△CT method.