Chamq universal sybr qpcr master mix q711

To verify the accuracy of the RNA-seq data, a total of 12 DEGs related to SI were selected and evaluated by qRT−PCR. The total RNA of the 21 samples in seven comparison groups (NPT0, CKT3, CKT1, SPT1, SPT3, CPT1, CPT3) was extracted by the Goldenstar™ RT6 cDNA Synthesis Kit Ver.2 (Beijing TsingKe Biotech Co., Ltd, China), and the remaining elimination of potential gDNA and reverse transcription were performed with HiScript^® II Q RT SuperMix for qPCR (+gDNA wiper) (Vazyme, Nanjing, China). Specific primers for the 12 DEGs were designed by Primer Premier 5.0 (Supplementary Table S1). The qRT−PCR was conducted in conjunction with ChamQ Universal SYBR qPCR Master Mix #Q711 (Vazyme, Nanjing, China). The experiment was performed on the CFX96 Real Time PCR System (Bio-Rad, Hercules, CA, USA), and CoGAPDH (KC337052.1) was chosen as the reference gene for Camellia oil tree. All analyses were repeated with three biological replicates, and the relative expression level of 12 DEGs were calculated with the 2^-ΔΔCt method. The results were analyzed using Microsoft Office Excel 2013 (Microsoft, Redmond, WA, USA) and SPSS (ver. 20.0; SPSS, Inc., Chicago, IL, United States) software.

A total of nine DEGs associated with the LSI of the Camellia oil tree were chosen and assessed by qRT-PCR in order to confirm the correctness of the RNA-seq results. A plant RNA kit (OMEGA, New York, NY, USA) was used to extract the RNA from those samples, and HiScript^® II Q RT Su-perMix for qPCR (+gDNA wiper) (Vazyme, Nanjing, China) was used to reverse transcribe and remove the potential remaining gDNA. By using Primer Premier 5.0, specific primers for the 9 DEGs were designed (Supplementary Table S4). The ChamQ Universal SYBR qPCR Master Mix #Q711 (Vazyme, Nanjing, China) was used in the QRT-PCR procedure. CoGAPDH was selected as the reference gene for the Camellia oil tree in this experiment, which was carried out on a CFX96 Real Time PCR System (Bio-Rad, Hercules, CA, USA). Using the 2^−∆∆CT method, the target gene’s expression level was determined. Three duplicates of each sample were examined.

To verify the reliability of the transcriptome results, 8 DEGs co-expressed in B116-CK, B116-T, B144-CK, and B144-T were selected, and the primers were designed using SnapGene 3.2.1 (the sequences are listed in Supplementary Table S1) and were synthesized by Tsingke Biotechnology Co., Ltd. (Beijing, China) before then being synthesized using the ChamQ Universal SYBR^® qPCR Master Mix Q711 (Vazyme Biotech Co., Ltd., Nanjing, China). The RNA used for quantitative RT-PCR was the same as that used to construct the cDNA library, and the related genes were normalized to Actin transcript levels by the 2^−ΔΔCt method [37 (link)].