Identification of endogenous reference genes for RT-qPCR analysis of plasma microRNAs levels in rats with acetaminophen-induced hepatotoxicity
Identification of endogenous reference genes for RT-qPCR analysis of plasma microRNAs levels in rats with acetaminophen-induced hepatotoxicity
复制标题
内源性参考基因的鉴定，用于对对乙酰氨基酚诱导的肝毒性大鼠血浆 microRNA 水平进行 RT-qPCR 分析
DOI：
10.1002/jat.2864
复制
发表时间：
2013-11-01
影响因子：
3.3
通讯作者：
Ma, Jing
中科院分区：
文献类型：
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作者：
Wang, Yan;Tang, Naping;Ma, Jing
循环microRNA（miRNA）表达谱已被报道是临床前和临床实践中药物诱导的肝损伤的有希望的生物标志物。正确的标准化对于准确的miRNA表达分析至关重要。本研究采用SYBR绿色实时荧光定量PCR（RT-qPCR）技术，对醋氨酚肝毒性大鼠血浆中常用的小RNA（U6，5S）和小RNA（let-7a，miR-92 a，miR-103和miR-16）等6个候选参考基因的表达稳定性进行了研究。使用geNorm、Normfinder、BestKeeper和比较delta-Ct统计模型分析数据，结果一致显示miR-103是最稳定表达的参考基因。而常用的管家基因5S或U6都不是合适的标准化物，因为5S在表达中表现出广泛的变异性，而U6在血浆样品中具有低表达水平。然后评估参考基因对血浆miR-122标准化的影响;当标准化为最稳定的参考基因时，对乙酰氨基酚处理组和媒介物组之间存在显著差异。然而，当将数据标准化为较不稳定表达的基因miR-16时，获得了偏倚结果。因此，我们建议将miR-103作为对乙酰氨基酚诱导的肝损伤的血浆miRNAs分析的合适参考基因。本文提供的数据是至关重要的成功的生物标志物的发现和验证的早期阶段的对乙酰氨基酚肝毒性的诊断。版权所有（c）2013约翰威利父子有限公司
Circulating microRNA (miRNA) expression profiles have been reported to be promising biomarkers for drug-induced liver injury in preclinical and clinical practice. Proper normalization is critical for accurate miRNAs expression analysis. Herein, using SYBR green quantitative real-time PCR (RT-qPCR), we evaluated the expression stability of six candidate reference genes including two commonly used small RNAs (U6, 5S) and four miRNAs (let-7a, miR-92a, miR-103 and miR-16) in plasma of rats with acetaminophen-induced hepatotoxicity. Data were analysed using geNorm, Normfinder, BestKeeper and comparative delta-Ct statistical models, and the results consistently show that miR-103 is the most stably expressed reference gene. Whereas the commonly used housekeeping genes 5S or U6 are all not suitable normalizers, because 5S exhibits extensive variability in expression and U6 has a low expression level across the plasma samples. Then the effect of reference genes on normalization of plasma miR-122 was assessed; when normalized to the most stable reference gene there were significant differences between the acetaminophen-treated group and the vehicle group. However, when the data were normalized to a less stably expressed gene, miR-16, a biased result was obtained. Therefore, we recommend that miR-103 as suitable reference gene for plasma miRNAs analysis for acetaminophen-induced liver injury. Data presented in this paper are crucial to successful biomarker discovery and validation for the diagnosis of the early stage of acetaminophen hepatotoxicity. Copyright (c) 2013 John Wiley & Sons, Ltd.