To detect mRNA expression by RT-qPCR, total RNA was purified with Trizol (Xi'an Chemical Reagent Factory), and then reverse-transcribed into cDNA by Superscript First-Strand Synthesis Kit (Invitrogen), followed by RT-qPCR with PrimeScript RT Master Mix (Cat# RR036A, TaKaRa, Tokyo, Japan). Primers were synthesized using TsingKe Biotechnology (Beijing, China). RT-qPCR was performed at 95 °C for 3 min, followed by 40 cycling cycles: 95 °C for 15 s, 60 °C for 15 s, 72 °C for 15 s, and finally melting curve analysis.
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