RNA Extraction and qRT-PCR Analysis

The total RNA was extracted from each sample using TRIzol reagent (Invitrogen, Carlsbad, CA, United States) according to the manufacturer’s instructions, then the RNA quality, purity, and concentration were measured by spectrophotometric absorbance measurement. The cDNA was further synthesized from 1 μg of total RNA using a PrimeScript RT^TM Reagent Kit (TaKaRa, Dalian, China), in accordance with the manufacturer’s instructions. The qRT-PCR analysis was conducted using 2 × SYBR Premix Ex Taq II (TaKaRa, Dalian, China). The reaction solution was prepared in a total volume of 12.5 μl containing 1 μl cDNA, 6.25 μl of 2 × SYBR Premix Ex Taq, 4.25 μl of ddH₂O, and 0.5 μl of each gene-specific primer (10 μM). For each sample, the analysis was conducted in triplicate and normalized to β-actin by the 2^{–Δ Δ Ct} method (Livak and Schmittgen, 2000). The control was set as one. The primers for qRT-PCR are summarized in Table 2.

Primers for qRT-PCR.