Approximately 2.5 µg of total RNAs (obtained by using an RNeasy mini-RNAsimple Kit, Tiangen Biotech, Beijing, China) were added in a reverse-transcriptase reaction to generate the first strand of cDNA (by using the Revert Aid First Strand Synthesis Kit, Thermo, Waltham, MA, USA). The synthesized cDNA was served as the template for qPCR, in the GoTaq®qPCR Master Mix (Promega, Madison, WI, USA), before being deactivated at 95°C for 10 min, and then amplified by 40 reaction cycles of being annealed at 95°C for 15 s and then extended at 60°C for 30 s. The final melting curve was validated to examine the amplification quality. Of note, the mRNA expression level of β-actin served as an optimal internal standard control, relative to other mRNA expression levels presented as fold-changes.
All the forward and reverse primers of those indicated genes were shown in Table S1.
All the forward and reverse primers of those indicated genes were shown in Table S1.