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.