Quantifying Gene Expression via qRT-PCR

Total RNA was extracted from different tissues using a modified CTAB method, as previously described [37 (link)]. RNA extractions from petals at different flower developmental stages were carried out utilizing the Trizol method according to the manufacturer’s introduction (TaKaRa, Dalian, China). The first-strand cDNA was synthesized using the PrimerScript Reverse Kit (TaKaRa, Dalian, China). The real-time PCR primer pair for HcJMT1 was designed using Primer Premier 5 (Table S1), and its amplification specificity was confirmed via agarose gel electrophoresis and melting curve analysis. The amplification efficiency and correlation coefficient of the primer were determined using the standard curve method with a dilution series of cDNA templates. Real-time PCR was performed on an ABI 7500 Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA) using SYBR Premix Ex Taq (TaKaRa, Dalian, China), as described previously [37 (link),38 (link)]. Three independent amplifications were performed for each sample. Previously validated HcACT and HcRPS genes were used as reference genes for samples in different tissues and petals at different developmental stages, respectively [37 (link)]. The relative expression of HcJMT1 was calculated using the 2^−ΔΔCt method [52 (link)]. Analysis of variance was conducted using SPSS 22 software with Tukey’s test (p = 0.05).