高级检索+

红豆杉TbAP2基因荧光定量PCR体系的建立及优化

Optimization of Fluorescent Quantitative Real-time PCR System of Taxus chinensis var. mairei

    摘要
  • 摘要:
    目的 建立稳定可靠的、适合检测红豆杉（Taxus L.）TbAP2基因表达量的荧光定量PCR实验体系。对于检测该物种中基因的组织特异性表达具有重要意义。
    方法 以曼地亚红豆杉细胞为试材，提取总RNA并反转录为cDNA，根据TbAP2基因序列设计多对引物，合成内参基因TBC41的引物，采用正交试验L9（34）方法分别筛选以上2个基因5 μL和10 μL小反应体系及20 μL常用体系中的最佳组合，并通过cDNA模板用量和引物用量等方面进行优化，以确保基因扩增效率在90%~105%之间。
    结果 本研究建立了TBC41和TbAP2基因在5、10、20 μL体系下的荧光定量最佳PCR反应体系，在优化后的5 μL体系下，加入Mix（2×）Universal 2. 5 μL，cDNA模板1.0 μL，正反引物共1.5 μL，内参基因TBC41和目的基因TbAP2的扩增效率均为94%；在优化后的10 μL下，加入Mix（2×）Universal 5 μL，cDNA模板1.2 μL，正反引物共1.3 μL，TBC41和TbAP2的扩增效率分别为95%和94%。在优化后的20 μL下，加入Mix（2×）Universal 10 μL，cDNA模板0.5 μL，正反引物共1.5 μL，TBC41和TbAP2的扩增效率分别为93%和99%，以上各扩增体系回归系数R2均大于0.980。
    结论 在以上3种反应体系下，内参基因和目的基因均具有接近100%的扩增效率，表明本研究成功建立了适合检测红豆杉TbAP2基因表达量的荧光定量PCR实验体系，并为红豆杉其它基因的表达研究提供参考。

     

    Abstract:
    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%, respectively. In the optimized 20μL system, the amplification efficiency of TBC41 and TbAP2 were 93% and 99%, respectively. The regression coefficient R2 in all the three amplification system were greater than 0.980.
    Conclusion All the reaction systems mentioned above show that the amplification efficiency of TBC41 and TbAP2 close to 100%, indicating that these detection program are suitable to investigate the TbAP2 gene expression by qRT-PCR method.

     

    • HTML全文
    • 参考文献(14)
    • 相关文章
    • 施引文献
  • 资源附件(0)

/

返回文章
返回