mRNA and microRNA quality control for RT-qPCR analysis
mRNA and microRNA quality control for RT-qPCR analysis
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DOI：
10.1016/j.ymeth.2010.01.010
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发表时间：
2010-04-01
期刊：
影响因子：
4.8
通讯作者：
Pfaffl, M. W.
中科院分区：
文献类型：
--
作者：
Becker, C.;Hammerle-Fickinger, A.;Pfaffl, M. W.
高质量的样品材料，即非降解或片段化的RNA，对于经典基因表达谱的重要性是有据可查的。因此，RNA质量的分析是制备RT-qPCR和微阵列分析等方法的有价值的工具。为了验证RNA的完整性，自动化毛细管电泳的使用是当今最先进的技术。根据最近出版的MIQE指南，这些PCR前的评估必须在科学出版物中明确记录，以增加实验透明度。RNA质量控制也可以集成到新应用的常规分析中，如microRNA（miRNA）表达的研究，因为对于影响miRNA分析的因素知之甚少。安捷伦科技公司为2100生物分析仪提供了一种新的芯片实验室应用，使其能够以绝对量[pg]和小RNA的百分比[%]定量miRNA。最近的结果表明，这种分析方法受到总RNA完整性的强烈影响。持续的RNA降解伴随着小RNA片段的形成，导致芯片上miRNA量的高估。已知总RNA完整性会影响RT-qPCR的性能以及mRNA表达谱分析中的定量结果。实际研究确定了miRNA基因表达谱的可比效果。使用合适的标准化方法可以部分减轻总RNA完整性的损害作用。(C)2010年爱思唯尔公司All rights reserved.
The importance of high quality sample material, i.e. non-degraded or fragmented RNA, for classical gene expression profiling is well documented. Hence, the analysis of RNA quality is a valuable tool in the preparation of methods like RT-qPCR and microarray analysis. For verification of RNA integrity, today the use of automated capillary electrophoresis is state of the art. Following the recently published MIQE guidelines, these pre-PCR evaluations have to be clearly documented in scientific publication to increase experimental transparency.RNA quality control may also be integrated in the routine analysis of new applications like the investigation of microRNA (miRNA) expression, as there is little known yet about factors compromising the miRNA analysis. Agilent Technologies is offering a new lab-on-chip application for the 2100 Bioanalyzer making it possible to quantify miRNA in absolute amounts [pg] and as a percentage of small RNA [%]. Recent results showed that this analysis method is strongly influenced by total RNA integrity. Ongoing RNA degradation is accompanied by the formation of small RNA fragments leading to an overestimation of miRNA amount on the chip. Total RNA integrity is known to affect the performance of RT-qPCR as well as the quantitative results in mRNA expression profiling. The actual study identified a comparable effect for miRNA gene expression profiling. Using a suitable normalization method could partly reduce the impairing effect of total RNA integrity. (C) 2010 Elsevier Inc. All rights reserved.