*2.6. Quantitative Real-Time PCR*

A total of nine genes were differentially expressed and subjected to qRT-PCR verification. Total RNA was extracted using the RC411-01 RT-PCR reagent (Vazyme Biotech, Nanjing, China). The cDNA was synthesized according to the instructions of the M-MLV reverse transcriptase Kit (28025013) (Thermo Fisher Scientific, Waltham, MA, USA). The qRT-PCR was FastStart Universal SYBR Green Master (Rox) superMIX reaction kit (4913850001) (Roche, Mannheim, Germany). The *Actin* gene was used as an internal reference. The primer pairs were designed using Primer Premier 5.0 software. The specific primers for genes involved in the hormone signaling pathway are listed in Table 1. Each biological sample was tested in triplicate, and the standard deviation (SD) values of the means were calculated using standard statistical methods. The expression of genes was analyzed using the DDCt data analysis method, and gene relative expression was calculated using the 2−ΔΔCt method.


**Table 1.** Primers for qRT-PCR expression analysis.

#### *2.7. Data Processing and Analysis*

We used Excel 2017 software for the data statistics, and the data drawing application Graphpad Prism 8.4.3. We used SPSS 21.0 (IBMCorp., Armonk, NY, USA) software to analyze the significant differences in the data based on the one-way analysis of variance (ANOVA) and Duncan methods, and the significant difference level was *p* < 0.05.
