RT-qPCR Validation of Grape RNA-seq DEGs

A total of 15 DEGs were randomly selected from the RNA-seq data to conduct the RT-qPCR analysis for further confirmation. RNA was extracted from 2 g frozen table grape tissue (stored at −80 °C) using a Spin Column Plant Total RNA Purification Kit (Sangon Biotech, Shanghai, China). Both the purity and quantity were checked using a spectrophotometer (Thermo Scientific, Waltham, MA, USA) at wavelengths of 260 and 280 nm, respectively. The quality of RNA was evaluated using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA). The first strand of cDNA was synthesized from the RNA using a PrimeScript RT reagent kit with a gDNA Eraser (Takara Biotechnology, Dalian, China) in a PCR System. Specific primers were obtained from Sangon Biotech (Shanghai, China) and are listed in Table 1. The RT-qPCR was conducted with a Bio-Rad CFX96 Real-Time PCR System (Applied Biosystems, Irvine, CA, USA) and the computer program was set according to Wang et al. [17 (link)]. RT-qPCR was carried out according to the method describe by Xu et al. [9 (link)]. The experiment was conducted twice, and there were three replications per treatment.