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Gene expression analysis by quantitative real-time PCR (qRT-PCR)

CL Chenglin Liang
TW Tian Wan
RW Rendun Wu
MZ Mei Zhao
YZ Yue Zhao
YC Yuliang Cai
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According to the manufacturer’s instructions, total RNA of root neck tissues was extracted using RNAprep Pure Plant Kit (Tiangen Biotech Co., Ltd., Beijing, China). The PrimeScript™RT Reagent Kit (Takara Biotechnology Co., Ltd., Dalian, China) was used to synthesize single-stranded cDNA. The SYBR Premix Ex Taq Kit (Takara Biotechnology Co., Ltd., Dalian, China) was used to perform qRT-PCR on a Life Technologies QuantStudio®5. The conditions of PCR were as follows: initial incubation at 95 °C for 30 s, followed by 40 cycles at 95 °C for 5 s and 60 °C for 30 s. The primers were designed by Primer Premier v5 software (Additional file 1: Table S2). ACTIN and YC-ACTIN served as an internal reference for normalizing gene expression in ‘CDR-1’ and tobacco plants, respectively. All qRT-PCR experiments were performed in triplicate using three biological replicates and three technical replications. Statistical significance was determined using Student’s t-test by IBM SPSS Statistics v21.0.

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