2.6.2 | RNA extraction and RT-qPCR analysis
Total RNA was extracted using TRIzol reagent, and the cDNA template was obtained by reverse transcription. The reaction for fluorescence quantitative PCR consisted of 12.5 μL of SYBR fluorescent dye, 1 μL of cDNA template, 0.5 μL of forward and reverse primers, and ddH2O to a final volume of 25 μL. The following PCR cycling condition was employed: 95 °C for 3 min, followed by 40 cycles of 95 °C for 10 s, 60 °C for 15 s, and 72 °C for 20 s. The relative gene expression values were calculated using the 2-ΔΔCTmethod (Livak & Schmittgen, 2001).
2.7|