Thunderbird qpcr mix qps 201

Total RNA of the above seven tissue samples was extracted by column plant RNAout kit (Tiandz, Beijing, China) according to the manufacturer’s instructions. About two µg total RNA was applied to synthesized first strand cDNA using reverse transcriptase AT311-03 (TransGen Biotech, Beijing, China). PCR primers (Table S1) of LcARFs and reference genes GAPDH and EF (Zhong et al., 2011 (link)) were designed by Primer Premier 5.0. qRT-PCR was performed according to the manufacturer’s specifications of THUNDERBIRD qPCR MIX QPS-201 (Toyobo, Shanghai, China) on a LightCycler 480 (Roche, Rotkreuz, Switzerland). Each expression profile was independently verified in three biological replicates. Relative expression level of each gene was calculated by the 2^−ΔΔCt method (Livak & Schmittgen, 2001 (link)). Significance analysis was conducted in SPSS version 22.0 and visualized by SigmaPlot 12.5.

Total RNA was extracted by TRIzol reagent (Invitrogen, Beijing, USA), and cDNA was synthesized using the AT311-03 cDNA kit (TransGen Biotech, Beijing, China) according to the manufacturer’s instructions. qRT‒PCR was carried out using THUNDERBIRD qPCR Mix QPS-201 (Toyobo, Shanghai, China) and an ABI 7500 Real-Time PCR System (Applied Biosystems, Waltham, CA, USA). The PCR program was as follows: 10 min at 95 °C, with 40 cycles of 15 s at 95 °C and 60 s at 55 °C. Statistical analysis was performed after obtaining the Ct values from three biological replicates. The UBQ5 gene was employed as the internal reference. The ∆∆Ct values were calculated and presented as the means ± standard errors (SE) of three replicates. The PCR primers utilized in this study are listed in Supplementary Table S6.