Quantitative Reverse Transcription PCR Analysis

The first-strand complementary DNA (cDNA) was synthesized from total RNA using PrimeScript™ RT Reagent Kit with gDNA Eraser (Takara, Beijing, China). qPCR was carried out using the ChamQ^TM SYBR^® qPCR Master Mix (Vazyme, Nanjing, China) and run on a CFX96™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) following the manufacturer’s instructions. The specific qPCR primers were designed using AlleleID 6 software (PREMIER Biosoft, Palo Alto, CA, USA) (Supplementary Materials, Figure S1), gene expression levels were normalized to the reference gene (28S rRNA) [35 (link)]. The qPCR programs were set as following: enzyme activation at 95 °C for 30 s, followed by 40 cycles with denaturation at 95 °C for 5 s, annealing at 60 °C for 30 s, and melting curve analysis. The mRNA expression levels were determined by the comparative 2^−△△CT method [36 (link)].