Primescript rt reagent qpcr kit with gdna eraser

In order to validate the DEGs identified from transcriptome sequencing, qPCR analysis was performed for two treatment groups (PCA vs. control). Total RNAs were extracted as described before to obtain the samples used in transcriptome sequencing. Using the PrimeScript RT reagent qPCR Kit with gDNA Eraser (Takara, Dalian, China), genomic DNA was removed from total RNA (300 ng RNA of each sample), and cDNA was synthesized. The PCR mixture contained 10 μl of EvaGreen 2 qPCR MasterMix-No dye [Applied Biological Materials (ABM) Inc. Vancouver, Canada], 7.4 μl of ddH₂O, 0.8 μl of each gene-specific primer (10 μM), and 1 μl of cDNA template. The qPCR assays were performed in an Applied Biosystems 7500 Real-Time PCR System (ABI, Waltham, MA, United States), with the following program: 95°C for 10 min, 40 cycles of 94°C for 15 s, and 60°C for 30 s. The actin gene was used to normalize the expression levels of the target genes. The relative expression levels of the target genes were calculated by the 2^–ΔΔCT method (Livak and Schmittgen, 2001 (link)). The primers were designed using Primer 3 software version 4.1.0⁵ and were synthesized by Sangon Biotech (Shanghai) Co., Ltd., Shanghai, China (Supplementary Table 1).

In order to validate the DEGs identified from transcriptome sequencing, quantitative real-time PCR (qRT-PCR) analysis was performed. Independent wild oil-tea camellia leaf samples at different air temperatures were used: JG05 at 17.8 °C and JG06 at 14.7 °C from Jinggang Mountain; LS05 at 11.0 °C and LS06 at 4 °C from Lu Mountain. Total RNAs were extracted as described before for the samples used in transcriptome sequencing. Using the PrimeScript™ RT reagent qPCR Kit with gDNA Eraser (Takara, Dalian, China), genomic DNA was removed from total RNAs (300 ng RNAs of each sample) and cDNA was synthesized. The PCR mixture contained 12.5 μL SYBR® Premix Ex Taq™ II (Tli RNaseH Plus) (Takara, Dalian, China), 9.5 μL ddH₂O, 1 μL of each gene-specific primer (10 μM) and 1 μL cDNA template. The qRT-PCR assays were performed in a CFX96 Touch™ RT-PCR Detection System (Bio-RAD, USA) with the following program: 94 °C for 2 min; 40 cycles of 94 °C for 20 s, 57 °C for 20 s and 72 °C for 30 s. A commonly used reference gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, was used to normalize the expression levels of target genes [32 (link)]. The relative expression levels of target genes were calculated with the 2^−∆∆Cq method [32 (link)].