RT-qPCR for Messenger RNA Quantification

The total RNA of each sample was treated with DNase I (Thermo Fisher Scientific, USA) to remove the genome. The synthesis of complementary DNA (cDNA) from 500 ng of total RNA was reverse transcribed by using the RevertAid™ Master Mix Kit (Thermo Fisher Scientific, USA). The primer sequence was referenced from the online website of PrimerBank (https://pga.mgh.harvard.edu/primerbank/index.html) and the specificity was verified by the primer basic local alignment search tool (BLAST) tool of the National Center for Biotechnology Information (NCBI) (Table 1). Gene expression was detected by RT-qPCR by using 2 × RealStar Green Fast Mixture [with Carboxy-X-rhodamine (ROX)] (GenStar, China) following the manual of the manufacturer. Data were collected and analyzed by using ABI QuantStudio™ version 5 software (Applied Biosystems, USA). The results were normalized to Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (human)/Gapdh (mouse) and relative expression was shown as 2^−ΔΔCt.

Sequences of the primer for quantitative real-time PCR (RT-PCR).