Quantitative Real-Time PCR Analysis

Total RNA was extracted using Total RNA Rapid Extraction Kit (Zomanbio, Beijing, China). First strand cDNA was synthesized using PrimeScript^@ Reverse Transcriptase (TaKaRa, Dalian, China). qRT-PCR was performed in a total volume of 20 μL reaction containing 100 ng of template cDNA, 0.2 μM of each primer, and 10 μL of 2 [SYBR premix Ex TaqTM (TaKaRa)]. The amplification program was as follows: one cycle of 30 s at 95°C, followed by 40 cycles of 5 s at 95°C, 34 s at 60°C. PpTEF2 (GDR accession no. ppa001368m) was selected as an internal control according to a previous report (Tong et al., 2009 (link)). The standard curve method was conducted to quantify the transcripts. All analyses were repeated three times. The sequences of primers used for real-time PCR analysis are listed in Supplementary Table S2.