Hiscript 2 reverse transcriptase based two stepqpcr kit

The pre-extracted
RNA was reverse-transcribed
into cDNA using a HiScript II Reverse Transcriptase-based two-step
qPCR kit (Vazyme Biotech Co. Ltd., Nanjing, China). Eight different
genes involved in flavonoid biosynthesis were selected for validation.
Primer5.0 software was used for primer design, and beta-Actin-1 was
selected as the internal reference gene.^{50 (link)} Three technical replicates were used for each gene involved in the
validation. Three biological replicates were also used for each group
of four samples at different developmental stages. The amplification
system was constructed using ChamQ Universal SYBR qPCR Master Mix
(Vazyme Biotech Co. Ltd., Nanjing, China) and placed in CFX Connect
(Bio-Rad Laboratories Inc. Hercules, CA, USA) for real-time fluorescence
quantitative PCR. The relative expression of genes was calculated
using the 2^–ΔΔCt method.
The Corrplot package in R-3.6.1 was used for correlation analysis
to verify the credibility of the transcriptome data analysis results.
The data were graphically plotted with Origin Pro software (version
8.0).^{46 (link),47 (link)}