Quantitative real-time PCR (RT‒PCR)
After total RNA extraction, cDNA was reverse transcribed with a
PrimeScript RT Reagent Kit (TaKaRa, Dojindo, Kumamoto, Japan) under the
following conditions: 15 min at 37 ℃; 5 s at 85 ℃; and an infinite hold
at 12 ℃. qPCR was performed with HieffTM qPCR SYBR ® Green Master Mix
(Yeasen Bio, Shanghai, China) in an ABI 7500 RT‒PCR System (Applied
Biosystems, Foster City, CA, USA). Amplification and detection were
performed under the following conditions: a 5-min hot start at 95 °C
during the holding stage; 40 cycles of 10 s at 95 °C, 30 s at 60 °C
during the cycling stage; and 15 s at 95 °C, 1 min at 60 °C, and 15 s at
60 °C during the melt curve stage. Relative gene expression was
calculated using the comparative 2-ΔΔCt method.
The primer sequences were as follows: