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

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.