TaqMan-based, real-time quantitative polymerase chain reaction method for RNA editing analysis
TaqMan-based, real-time quantitative polymerase chain reaction method for RNA editing analysis
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DOI：
10.1016/j.ab.2009.04.011
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发表时间：
2009-07-15
影响因子：
2.9
通讯作者：
Dracheva, Stella
中科院分区：
文献类型：
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作者：
Wong, Kevin;Lyddon, Rebecca;Dracheva, Stella
异常的腺苷-肌苷（A-to-1）信使RNA （mRNA）编辑与中枢神经系统的几种疾病状态有关。在这里，我们报告了一种基于TaqMan探针的定量聚合酶链反应（qPCR）来确定特定位点RNA编辑频率的实验。通过测量人盐酸盐受体GluR5亚基Q/R位点上A-to-1 mRNA的编辑频率，对该方法进行了测试，并与两种已建立的评估RNA编辑的方法进行了比较：单个克隆测序和限制性内切分析。qPCR检测显示出高灵敏度和可重复性，在编辑和未编辑的转录本变体之间表现出特殊的区别，并且证明与其他编辑方法相比具有几个优势。由于基于taqman的qPCR技术可以很容易地适应不同的编辑目标，这种新技术所提供的增强的能力应该有助于各种RNA编辑研究，旨在阐明这一过程在正常生理和疾病中的作用。Elsevier Inc.出版。Elsevier Inc.出版。
Abnormal adenosine to inosine (A-to-1) messenger RNA (mRNA) editing has been linked to several disease states afflicting the central nervous system. Here we report an assay to determine RNA editing frequencies at specific sites that is based on quantitative polymerase chain reaction (qPCR) with TaqMan probes. The assay was tested by measuring the frequency of the A-to-1 mRNA editing at the Q/R site of the human kainate receptor Subunit GluR5 and was compared with two established methods of assessing RNA editing: sequencing of individual clones and restriction analysis. The qPCR assay displayed high sensitivity and reproducibility, demonstrated exceptional discrimination between edited and Unedited transcript variants, and proved to have several advantages over the other editing methods. Due to the fact that TaqMan-based qPCR technology can be easily adapted to different editing targets, the increased capabilities afforded by this new technique should facilitate various RNA editing studies that aim to elucidate the role of this process in normal physiology and in disease. Published by Elsevier Inc. Published by Elsevier Inc.