RNA Extraction and RT-qPCR Analysis of Brassica napus

We extracted total RNA from various tissues of B. napus cultivar Zhongshuang11 (ZS11) using the EZ-10 DNAaway RNA Mini-prep Kit (Sangon Biotech (Shanghai), Co., Ltd). We then synthesized first-strand cDNAs from 1 µg total RNA using the PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa Biotechnology, Dalian, China) according to the manufacturer’s instructions. We performed real-time quantitative PCR analysis using SYBR Premix Ex Taq II (Perfect Real Time) (TaKaRa, Dalian, China) in a CFX96 real-time PCR system (Bio-Rad, USA) according to previous methods [28 (link)]. Gene-specific primers were designed using Vector NTI software, with the BnACTIN7 gene as internal reference gene (Additional file 1: Table S4) [7 (link)]. We performed RT-qPCR on three independent biological replicates, each consisting of three technical replicates. We determined BnaFAX1-1 transcript levels in WT plants and transgenic lines by RT-qPCR as described above. In Arabidopsis, we used ACTIN2 as an internal control (Additional file 1: Table S4) [45 (link)].