*2.4. RT-PCR and RT-qPCR Data Analysis*

To verify the accuracy of the designed primers, each pair was used for RT-PCR amplification. Each 20 μL reaction system contained 2 μL of 10 μM forward and reverse primers, 1 μL cDNA, 10 μL 2 × Rapid Taq Master Mix (Vazyme, Nanjing, China) and 7 μL ddH2O. The reaction was carried out at 95 ◦C for 3 min, followed by 35 cycles of 95 ◦C for 30 s, 60 ◦C for 30 s and 72 ◦C for 15 s, with a final extension at 72 ◦C for 2 min. The PCR products were visualized by 1% (*w*/*v*) agarose gel electrophoresis.

To monitor the E-value, the cDNA templates from all samples were serially diluted five-fold (cDNA:water, v:v). RT-qPCR was performed for each pair of primers to obtain Ct values and to establish a standard curve; the R2, slope and E-values were calculated with Microsoft Office Excel 2019 using the following formula: E = (5−1/slope − 1) × 100% [43].

All RT-qPCRs were performed with an Applied BiosystemsTM (ABI) QuantStudioTM 5 real-time PCR system (ABI, Los Angeles, CA, USA) using the following amplification procedure: 95 ◦C for 30 s, followed by 40 cycles of 95 ◦C for 5 s and 60 ◦C for 30 s. A melting curve was generated at 60–95 ◦C. Each 20 <sup>μ</sup>L RT-qPCR reaction (10 <sup>μ</sup>L 2 × SYBR® Green *Pro Taq* HS Premix, 2 μL 5-fold diluted cDNA, 0.4 μL 10 μM forward primer, 0.4 μL 10 μM reverse primer, 0.4 μL 4 μM ROX Reference Dye, and 6.8 μL RNase-free water) was prepared according to the instructions for SYBR® Green Premix Pro Taq HS qPCR Kit (Rox Plus) (Accurate Biology, Shanghai, China). A negative control without the addition of cDNA was used to test for background amplification. Three technical replicates were performed for each sample, and the mean was used for RT-qPCR analysis.
