Digital Quantitation of Potential Therapeutic Target RNAs
Digital Quantitation of Potential Therapeutic Target RNAs
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DOI：
10.1089/nat.2013.0427
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发表时间：
2013-06-01
影响因子：
4
通讯作者：
Corey, David R.
中科院分区：
文献类型：
--
作者：
Dodd, David W.;Gagnon, Keith T.;Corey, David R.
准确测定细胞内给定RNA的量对于充分了解RNA的功能和调节是必要的。通常，RNA的丰度是通过定量聚合酶链式反应(QPCR)来测量的。然而，对于定量聚合酶链式反应，除非产生足够的参考标准曲线，否则绝对定量是不可能的。该方法不太适合检测低拷贝数模板，其值因所用的特定引物而异。为了克服这些缺点，发展了数字聚合酶链式反应(DPCR)来获得样本中RNA拷贝的精确值。在这里，我们报告了液滴数字聚合酶链式反应(DdPCR)的特征。我们使用ddPCR对来自人类细胞内不同亚细胞隔间的长非编码RNA进行了量化，发现使用ddPCR获得的结果与qPCR获得的结果相似。突变的亨廷顿蛋白(HTT)是亨廷顿病的病因，我们证明了我们可以定量人类HTT信使RNA，并使用ddPCR区分突变和野生型HTT等位基因。这些结果揭示了使用ddPCR的实验设计的洞察力，并表明ddPCR可以作为一种强大的工具来识别细胞内RNA物种的数量。
Accurate determination of the amount of a given RNA within a cell is necessary to gain a full understanding of the RNA's function and regulation. Typically, the abundance of RNA is measured by quantitative polymerase chain reaction (qPCR). With qPCR, however, absolute quantification is not possible unless an adequate reference standard curve is generated. The method is not well suited for detecting low copy number templates and values vary depending on the specific primers used. To overcome these drawbacks, digital PCR (dPCR) has been developed to obtain exact values for RNA copies in a sample. Here we report the characterization of droplet digital PCR (ddPCR). We used ddPCR to quantify long noncoding RNAs from various subcellular compartments within human cells and found that results obtained using ddPCR parallel those from qPCR. Mutant huntingtin (HTT) protein is the cause of Huntington's Disease, and we show that we can quantify human HTT messenger RNA and discriminate between the mutant and wild-type HTT alleles using ddPCR. These results reveal insights into the design of experiments using ddPCR and show that ddPCR can be a robust tool for identifying the number of RNA species inside of cells.