Quantification of miRNA and mRNA Expressions

Total RNA was extracted from the cortex of rats and HEK293 cells using the Ultrapure RNA Kit (CWBIO, Beijing, China). miRNA 1st Strand cDNA Synthesis Kit and miRNA Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) were used to measure the expression of DEMs in the 2VO rat model. Likewise, the DEGs in the rats with 2VO were also measured using Hiscript^®III RT SuperMix for qPCR and ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China). Quantitative real-time polymerase chain reaction (qRT-PCR) analyses for miRNA and mRNA were performed following the manufacturer’s recommendations. The expression of miRNA and mRNA was calculated by the 2^–ΔΔCT method (Yi et al., 2021 (link)). The primes in this study were synthesized from Sangon Biotech (Shanghai, China) and are listed in Table 1. U6 small RNA was used as the internal reference for miRNA. Gapdh and ACTB were used as the reference genes for mRNA.