基于芯片数据的烟草qrt-pcr内参基因鉴定与验证-烟草科技.pdfVIP

2015 年2 月 烟草科技 Feb. 2015 第48 卷第2 期 Tobacco Science Technology Vol. 48 No. 2 基于芯片数据的烟草qRT-PCR 内参基因鉴定与验证 * 王 燃，许亚龙，李泽锋，卢 鹏，孟利军，曹培健 中国烟草总公司郑州烟草研究院，郑州高新技术产业开发区枫杨街2 号 450001 摘要：实时荧光定量PCR （qRT-PCR ）结果的准确性关键在于选择合适的稳定内参基因，而常 用持家基因的表达不是恒定不变的，无法满足qRT-PCR 准确定量的要求。为发掘烟草中优于 传统持家基因的新内参基因，分析了烟草100 张基因芯片数据，鉴定了稳定表达的候选内参基 因，并利用烟草各个发育时期的RNA 样品，通过qRT-PCR 实验比较并验证了其表达的稳定 性。结果显示, 新鉴定的烟草内参基因HSC 70-1 表达稳定性优于所有的常用持家基因， HSC 70-1 ，L25 ，EF-1α和HIST2H3A 为烟草qRT-PCR 实验中的最优内参基因组合，同时使用4 个基因进行校正和标准化获得了可靠的定量结果。 关键词：烟草；实时荧光定量PCR ；内参基因；芯片 中图分类号：TS413 文献标志码：A 文章编号：1002-0861（2015 ）02-0001-06 DOI: 10.16135/j.issn 1002-0861 IdentificationandValidationofTobaccoReferenceGenesforqRT-PCRBased onMicroarrayData WANG Ran, XU Yalong, LI Zefeng, LU Peng, MENG Lijun, and CAO Peijian* Zhengzhou Tobacco Research Institute of CNTC, Zhengzhou 450001, China Abstract: The accuracy of Real Time Fluorescent Quantitative RT-PCR (qRT PCR) lies on - - the selection of reference genes. Conventional housekeeping genes due to not always consistent in expression are not applicable for qRT PCR. In order to find out new tobacco - reference genes, the data of 100 Affymetrix microarrays were analyzed, and candidates for reference gene were identified. Their expression was compared and validated with the RNA samples of tobacco leaves of different developmental stages by qRT PCR. The results - showed that the new-identified reference gene HSC 70 1 had the advantage over all c