RNA extraction and Real-time quantitative RT-PCR were conducted as described previously [71 (link)]. Total RNA was extracted from the samples using a TRIzol reagent kit (Invitrogen, Carlsbad, CA, US) according to the manufacturer’s specifications. The RNA integrity was evaluated using agarose gel electrophoresis and ethidium bromide staining. The RNA preparation was then treated with Dnase I and first strand synthesis of cDNA was performed by using oligo (dT) primer and RT Enzyme (Thermo Fisher, USA).
The quantitative real-time PCR was carried out with SYBR-green fluorescence using a CF × 96 Real Time System (BIORAD) with a 20 μl PCR reaction mixture that included 8.8 μl of diluted cDNA, 10 μl of 2 × FastStart Universal SYBR Green Master (ROX) (Roche, Switzerland), and 0.6 μl of forward and reverse primer (Additional file9 ). The BraA02g003190 gene was used as a reference gene. Each sample was run in triplicate for analysis. At the end of the PCR cycles, melting curve analysis was performed to validate the specific generation of the expected PCR product. The expression levels of RR-TZF genes were calculated with the 2 − ΔΔCT method [78 (link)].
The quantitative real-time PCR was carried out with SYBR-green fluorescence using a CF × 96 Real Time System (BIORAD) with a 20 μl PCR reaction mixture that included 8.8 μl of diluted cDNA, 10 μl of 2 × FastStart Universal SYBR Green Master (ROX) (Roche, Switzerland), and 0.6 μl of forward and reverse primer (Additional file
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