生物技术通报 ›› 2017, Vol. 33 ›› Issue (11): 174-179.doi: 10.13560/j.cnki.biotech.bull.1985.2017-0352

• 研究报告 • 上一篇    下一篇

华细辛实时荧光定量PCR分析中内参基因的选择

赵晓冰1,2, 潘磊2, 柳威2, 林懋怡2, 李宏庆1, 刘忠2   

  1. 1. 华东师范大学生命科学学院,上海 200241;
    2. 上海交通大学药学院,上海 200240
  • 收稿日期:2017-05-03 出版日期:2017-11-26 发布日期:2017-11-22
  • 作者简介:赵晓冰,女,硕士研究生,研究方向:生物学,E-mail:1194089861@qq.com;潘磊同为本文第一作者
  • 基金资助:
    国家自然科学基金项目(81373962,31570325)

Screening of Reference Genes for Quantitative Real-time PCR Analysis in Asarum sieboldii

ZHAO Xiao-bing1,2, PAN Lei2, LIU Wei2, LIN Mao-yi2, LI Hong-qing1, LIU Zhong2   

  1. 1. School of Life Science,East China Normal University,Shanghai 200241;
    2. School of Pharmacy,Shanghai Jiao Tong University,Shanghai 200240
  • Received:2017-05-03 Published:2017-11-26 Online:2017-11-22

摘要: 选择合适的内参基因是准确分析目标基因表达水平变化的重要条件。选取SAND-1、ACT、18SrRNA、CYP2、GAPDH、TUB 6个常用的内参基因作为候选内参基因,利用实时荧光定量PCR技术,通过geNorm、NormFinder、BestKeeper、Delta CT等软件分析和评价6个候选内参基因在华细辛营养生长期、花期和花后期等不同生长阶段的根、根茎、叶片、叶柄等不同组织中的表达稳定性,筛选适宜华细辛不同生长阶段不同组织基因表达水平分析的内参基因。结果表明,18S rRNA在华细辛所有样品中表达最为稳定,是进行华细辛基因表达水平分析的适宜的内参基因。研究结果可为华细辛重要活性成分生物合成途径相关基因的功能分析,以及基因差异表达的研究提供有效的校正工具,确保基因表达分析结果的准确性与可靠性。

关键词: 华细辛, 内参基因, geNorm, NormFinder, BestKeeper, Delta CT

Abstract: Selecting appropriate reference gene is key step for the accurate analysis of the expression variations of target genes. Using several common-used reference genes of SAND-1,ACT,18S rRNA,CYP2,GAPDH and TUB as candidate genes and quantitative real-time PCR analysis of gene expression,the expression stabilities of these 6 candidate genes in various samples from roots,rhizomes,leaves,petioles and flowers in different growth stages of nutrition growth,flowering and post-flowering were assessed,with a set of approaches such as geNorm,NormFinder,BestKeeper and Delta CT,representatively. It is for screening the appropriate reference genes in analysing gene expressions of Asarum sieboldiiMiq. in varied tissues at different growth stage. The results demonstrated that 18S rRNA was the optimal object to act as a proper reference gene in this species because it displayed the most stable expression in almost all samples. This study will provide a feasible and valid calibrate not only for gene function characterization concerning chemical compound biosynthesis but also for gene differential expression analysis in A. sieboldii,ensuring the accuracy and reliability of results.

Key words: Asarum sieboldii, reference gene, GeNorm, NormFinder, BestKeeper, Delta CT