Optimization of Fluorescent Quantitative Real-time PCR System of Taxus chinensis var. mairei / 红豆杉TbAP2基因荧光定量PCR体系的建立及优化

• Published in: Forest research (Beijing) Vol. 32; no. 1; p. 39
• Main Authors: 张恺恺, 吕星, 杨立莹, 陈段芬, 邱德有, 杨艳芳
• Format: Journal Article
• Language: Chinese
• Published: Beijing Chinese Academy of Forestry 01.01.2019
• Subjects: Efficiency; Gene expression
• ISSN: 1001-1498
• DOI: 10.13275/j.cnki.lykxyj.2019.01.006

[Objective] To establish a stable and suitable real-time fluorescent quantitative PCR (qRT-PCR) experiment system of TbAP2 in Taxus L. [Method] Total RNA was extracted from the cell line of Taxus×media and used to reverse transcript cDNA. According to the sequence of TbAP2 gene obtained previously, 7 primer pairs were designed and synthesized with the TBC41 gene as the housekeeping gene. The orthogonal test L9 (34) method were used to choose the stable and suitable qRT-PCR experiment system with the cDNA as template. The volume of qRT-PCR reaction included 5, 10 and 20μL. The amplification efficiency would be assured between 90%-105% by adjusting the dosage of cDNA template and primer pairs, respectively. [Result] This study established the optimal qRT-PCR reaction system of TBC41 TbAP2 and gene in 5μL, 10μL and 20μL volume. In the optimized 5μL system, the amplification efficiency of both TBC41 and TbAP2 were 94%. In the optimized 10μL system, the amplification efficiency of TBC41 and TbAP2 were 95% and 94%,