Quantitative Analysis of HvZF-HD Gene Expression

SteadyPure Universal RNA Extraction Kit (Accurate Biology) was used to isolate total RNA, and the first-strand cDNA was synthesized by using the Evo M-MLV kit with gDNA clean for qPCR II (Accurate Biology). The specific primers of HvZF-HD and HvActin genes were showed in Table S1 [26 (link)]. Real-time PCR experiments were conducted using TransTaq-T DNA Polymerase (TransGen, Beijing, China) under the following cycling program: initial denaturation at 95 °C for 3 min, followed by 40 cycles of denaturation at 95 °C for 5 s and annealing/extension at 60 °C for 30 s. Real-time PCR experiments were performed in three independent biological replicates and three technical replications to determine the average Ct values. The relative expression levels of HvZF-HD genes were calculated by 2^−∆∆CT method [27 (link)].