2.8 Quantitative real-time PCR analysis
The leaves of C. sinensis var. sinensis were used for gene
expression analysis. The tea samples were ground into powder under
liquid nitrogen and collected in an RNA-free centrifuge tube for RNA
extraction. Total RNA from tea plant leaves was used as a template.
Fluorescent quantitative cDNA templates were synthesized by reverse
transcription using Hifair® III First-Strand cDNA Synthesis SuperMix for
qPCR (gDNA digester plus) kit. Real-time PCR was performed according to
previously published protocols (Chen et al., 2020c) using the
gene-specific primer sequences (Table S1). The
glyceraldehyde-3-phosphate dehydrogenase (GAPDH ) gene was used as
an internal reference gene, and relative expression levels were
calculated using the 2–ΔCT method (Jing et al.,
2020). All reactions were carried out using the CFX96™ Real-Time System
(Bio-Rad, USA). The two-step temperature program was as follows: 95°C
for 3 min, followed by 40 cycles of 95°C for 10 s and 62°C for 30 s in
96-well optical reaction plates.