*2.6. Quantitative Real-Time PCR*

Four genes that were significantly differentially expressed were selected for QRT-PCR analysis, and GAPDH was used as the reference gene. The primers were designed with Primer-BLAST http://www.ncbi.nlm.nih.gov/tools/primer-blast/ (accessed on 3 November 2020) and are presented in Supplementary Table S1. Total RNA was reversetranscribed using the PrimeScript RT Reagent Kit with gDNA Eraser. The reactions were prepared on a StepOne PlusTM Real-time PCR detection system (ABI, Boston, MA, USA) with a total volume of 10 μL: 3 μL of 1:2 diluted template, 1 μL of each primer (5 μM), and <sup>5</sup> <sup>μ</sup>L of 2× Fast SYBR® Green Master Mix (ABI, Boston, MA, USA). Baseline, threshold cycles (Ct), and statistical analyses were automatically determined using the StepOne PlusTM Software version 2.3 (ABI, Boston, MA, USA).
