Stepone real time pcr machine

RNA was purified using GeneJET RNA Purification Kit (thermo scientific) and qPCR was performed on a StepOne real-time PCR machine (BIO-RAD) using SYBR Green PCR master mix (Promega). mRNA level of actin was used as control. Primers used for qPCR analysis are listed in Supplementary Table S8.

For B. napus and B. nigra samples, TRIzol reagent (Invitrogen, Germany) was used to extract total RNA in accordance with the manufacturer’s instructions, and the first cDNA strand was synthesized using a TaKaRa reverse transcription system (Japan) in accordance with the manufacturer’s protocol. For B. oleracea samples, RNA extraction kits (Omega, USA) were utilized in accordance with the manufacturer’s instructions. qRT-PCR was performed with Primers for qRT-PCR (Additional file 1: Table S1) were designed by Primer version 5.0. A qRT-PCR mixture was 15 μL in volume and composed of 7.5 μL of SYBR Green Master Mix reagent (Toyobo, Japan), 0.6 μL of specific primer, 2 μL of cDNA, and 5.9 μL of ddH₂O. qRT-PCR was run using a StepOne real-time PCR machine (BioRAD, USA) programmed to heat for 30 s at 95 °C, followed by 40 cycles of 5 s at 95 °C and 45 s at 55 °C–58 °C. The specificity of the reactions was verified through melting curve analysis. GAPDH [61 (link)] and 25S [62 (link)] were used as internal controls. Comparative ΔΔ^CT method was applied to analyze the relative expression levels of CRFs. Hierarchical clustering and heatmap representation of the expression pattern of CRFs were drawn in HemI [63 (link)]. Three biological replicates were included for each sample.