RNA质量对内参基因选择和qRT-PCR结果评价的影响  

Effects of RNA Quality on the Selection of Internal Reference Genes and Evaluation of qRT-PCR Results

作　　者：霍雪萍[1] 张琳萍[2] 赵向绒[1] 刘勤社[3] 王海芳 HUO Xue-Ping;ZHANG Lin-Ping;ZHAO Xiang-Rong;LIU Oin-She;WANG Hai-Fang(Laboratory Center of Shaanxi Province People＇s Hospital, Xi＇an 710068;Department of Integrated Traditional Chinese and Western Medicine, Medical School of Xi＇an Jiaotong University, Xi＇an 710061;Shaanxi Key Laboratory of Integrated Traditional and Western Medicine for Prevention and Treatment of Cardiovascular Diseases, Shaanxi University of Chinese Medicine, Xi＇an 712046 China)

机构地区：[1]陕西省人民医院中心实验室,陕西西安710068 [2]西安交通大学医学部,陕西西安710061 [3]陕西省中西医结合心血管病防治重点实验室,陕西中医药大学,陕西西安712046

出　　处：《生物技术通讯》2018年第3期415-418,共4页Letters in Biotechnology

基　　金：国家自然科学基金(81573823);陕西省中医管理局平台项目(JCPT028)

摘　　要：目的:研究RNA质量对内参基因选择和实时定量PCR(qRT-PCR)分析肿瘤坏死因子α(TNFα)诱导基因表达结果评价的影响。方法:用TNFα刺激体外培养的小鼠血管内皮细胞,采用qRT-PCR技术,以2种常用的管家基因β-actin和GAPDH为内参,检测细胞间黏附分子1(ICAM-1)基因的表达,探讨RNA纯度对TNFα下游基因表达检测结果的影响。结果:当提取的总RNA纯度很高(D260nm/D230nm>2.0)时,以β-actin和GAPDH为内参基因检测ICAM-1基因表达的结果相近;当总RNA可能被盐及小分子污染(D260nm/D230nm<2.0)时,以β-actin为内参基因检测的ICAM-1基因相对表达量显著高于以GAPDH为内参所得结果。结论:RNA质量显著影响荧光定量PCR对基因表达的检测结果,因此在RNA质量较低时应选择2个或以上内参基因进行校正,以减少实验结果误差,得到准确结果。Objective： To investigate the influence of RNA quality on the evaluation of tumor necrosis factor-α（TNF-α）-induced gene regulation by quantitative real-time PCR（qRT-PCR） and the selection of internal reference genes. Methods： The vascular endothelial cells（VECs） cultured in vitro were stimulated with TNF-α. qRTPCR was performed to measure the expression of intercellular cell adhesion molecule-1（ICAM-1） gene using two commonly used housekeeping genes β-actin and GAPDH as internal controls and the effect of RNA quality on the evaluation results was analyzed. Results： When the extracted total RNA D260 nm/D230 nm2.0, which means high the RNA purity, the results of ICAM-1 gene expression using β-actin or GAPDH as internal reference genes were close. However, when D260 nm/D230 nm2.0, which means contaminated RNA, the relative expression level of ICAM-1 gene normalized by β-actin was significantly higher than that normalized by GAPDH. Conclusion： The evaluation of gene expression by qRT-PCR is significantly affected by the quality of RNA. Therefore, when the RNA quality is low, two or more internal reference genes should be used for the correction of qualitative analysis.

关 键 词：RNA纯度 实时荧光定量PCR 内参基因 肿瘤坏死因子Α 

分 类 号：Q78[生物学—分子生物学]