RNA Extraction and Quantitative Real-Time PCR (qPCR)

Total RNA was separated and extracted by the RNeasy Mini Kit according to the manufacturer’s protocol (Vazyme Biotech, Nanjing, China). Then, 1.0 μg RNA was used as a template for cDNA synthesis by using the HiScript III 1^st Strand cDNA Synthesis Kit (+ gDNA wiper) (Vazyme Biotech). The cDNA samples were analyzed by qPCR using the AceQ qPCR SYBR Green Master Mix (Low ROX Premixed). U6 snRNA was used as an internal control. The experimental data were analyzed by the relative quantification method (2-∆∆Cq) [19]. All experiments were repeated thrice.