Easyspin plus total rna extraction kit

Total RNA of all collected samples was extracted using the EASYspin Plus Total RNA Extraction Kit (Aidlab, Beijing, China) according to the manufacturer protocol. The NanoDrop 2000 Spectrophotometer was used to determine the quantity and quality of RNAs. Subsequently, using the PrimeScript^TM 1st Strand cDNA Synthesis Kit (TakaRa, Dalian, China) to synthesize first-strand cDNAs. The specific primers were designed according to CDSs (Supplementary Table S3). All melting curves produced by qRT-PCR were shown in Supplementary Fig. S3.
The amplification reactions of qRT-PCR were performed with Lightcycler 96 system (Roche) using SYBR the premix Ex taq (TakaRa) in 20 μL volume with following parameters: initializing denaturation at 95 °C for 30 seconds, following 45 cycles denaturation at 95 °C for 10 seconds; annealing at 53–54 °C for 10 seconds, and extension at 72 °C for 20 seconds. In addition, the default setting for the melting curve stag was chosen. Three biological replicates were maintained for each collected sample. The relative expression levels were calculated according to the 2^−ΔΔCt method47 (link). The heatmap for expression profiles were generated with the Mev 4.0 software48 with Pearson correction and complete linkage clustering.

Total RNA was extracted from all samples using the EASYspin Plus Total RNA Extraction Kit (Aidlab, Beijing, China), and first-strand cDNAs were synthesized with the PrimeScript 1st Strand cDNA Synthesis Kit (TakaRa, Dalian, China) according to the manufacturer’s protocols. For quantitative real-time RT-PCR (qRT-PCR) assay, gene-specific primers were designed for the membrane-bound FAD genes according to their CDSs (S2 Table). The qRT-PCR was performed with the SYBR Premix Ex Taq (TakaRa, Dalian, China) in the BioRad CFX96 Real-time PCR System following the manufacturer’s instructions. The cotton UBQ7 gene was used as an internal reference for all the qRT-PCR analyses. Each sample was performed in three biological replicates. The relative expression levels were calculated according to the 2^-ΔΔCT method [39 (link)]. The expression profiles were clustered using the Cluster 3.0 software [40 (link)].

Total RNA was extracted using the EASYspin Plus Total RNA Extraction Kit (Aidlab Biotechnologies Co.). Genomic DNA was removed from total RNA by DNase (Thermo Fisher Scientific Inc.), and cDNA was synthesized by ReverAid First Strand cDNA Synthesis Kit (Thermo Scientific). The β‐tubulin gene was selected as the internal reference gene to determine the relative expression of target genes, and the fragment of 157 bp was amplified by primer pair NF and NR according to the cDNA sequence of β‐tubulin in Bmpc7 (Ma et al., 2003; Schmittgen and Livak, 2008). The primer pairs for RT‐qPCR are given in Table S1. Expression of the MfOfd1 gene was detected by RT‐qPCR with the primer pair MfF/MfR. RT‐qPCR was performed in a CFX96 Real‐Time PCR detection system (Bio‐Rad) using SYBR Green I fluorescent dye (Aidlab) in 20 μl volumes with 2 μl cDNA and 0.5 μl of each primer (10 μM). The experiments were performed with three independent biological repeats. The expression of the MfOfd1 gene was normalized to the expression of the β‐tubulin gene, and relative gene expression was calculated with the comparative C_t (2^−ΔΔCt) method (Wong and Medrano, 2005).