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RNA extraction and quantitative real-time PCR

HZ Haiyan Zhang
XZ Xiaohui Zhang
HZ Huixia Zhao
JH Jin Hu
ZW Zhaoyang Wang
GY Guangsheng Yang
XZ Xianming Zhou
HW Heping Wan
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Total RNA was extracted using the RNA simple Total RNA kit (Tiangen Biotech, Beijing, China) according to the manufacturer’s protocol. cDNA was synthesized with 1 µg RNA from each sample with HiScript® II Q Select RT SuperMix with gDNA wiper (Vazyme, Nanjing, China). Gene-specific primers used for quantitative real-time PCR (qRT-PCR) listed in Supplementary file 5. qRT-PCR was run on the AriaMx real-time PCR system (Agilent Technologies). The following cycling parameters were used: initial denaturation at 95 °C for 5 min; 40 amplification cycles consisting of denaturation at 95 °C for 10s, annealing and extension at 60 °C for 30 s; The melting curve was then tested at 65–95 °C. The internal standard was the B. napus actin gene (BnaA01g27090D). Three biotic replicates were performed for each sample, and each replicate contained three technical replicates. Relative expression levels were calculated according to the 2−ΔΔCt method [51].

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