Gene Expression Analysis by RT-qPCR

Gene primer sequences were designed using Primer-BLAST of the National Center for Biotechnology Information [110 (link)]. RNA was extracted using the TRIzol method. Reverse transcription and PCR amplification were performed using the One Step TB Green PrimeScript RT-PCR Kit (Takara). RT-qPCR was performed on LightCycler 96 (Roche, Basel, Switzerland), a real-time PCR system. Use NormFinder software (version 5, Aarhus University Hospital, Skejby, Denmark) to select the most stable gene pairs from the 6 genes as reference genes for RTqPCR assay [111 (link)]. The primer amplification efficiency was calculated by establishing a standard curve. Use the geometric mean of two carefully selected reference genes (GAPDH and MRPL39) as an accurate normalization factor [112 (link)]. Relative gene expression calculated by the modified Pfaffl method [8 (link),113 (link)]. The sequences and amplification efficiencies of the gene primers are provided in Table S1.