Quantification of Gene Expression via RT-qPCR

Total RNA was extracted using the Trizol method [71 (link),72 (link)]. All samples were tested for the concentration, A260/A280 ratio, and A260/A230 ratio by a Thermo SCIENTIFIC spectrophotometer. HiScript^® III RT SuperMix (Vazyme, Nanjing, China) was used to remove residual genomic DNA contamination and reverse transcription to obtain cDNA for qPCR. The Real-Time RT-PCR was carried out with a Roche Lightcyler^® 480 instrument using SYBR qPCR Master Mix in 20 μL volumes. The stable and reliable housekeeping TKS β-actin gene and GAPDH gene were used as internal controls. The Real-Time RT-PCR primers were designed by Primer Premier software (version 5, Premier, Vancouver, BC, Canada) (Table S6). The standard amplification procedure of the Real-Time RT-PCR was as follows: first, the holding stage is the reaction at 95 °C for 30 s, followed by the cycle stage, which runs 40 cycles at 95 °C for 5 s and 60 °C for 34 s, and finally the melting curve stage. The expression level was expressed as a change in relative multiples of 0 h (control) set to 1. Each reaction was performed in three biological replicates and the Real-Time RT-PCR data were analyzed by 2^−ΔΔCT method. The bar graph was drawn by GraphPad Prism (version 8.0.2, GraphPad Software, San Diego, CA, USA).