Mitochondrial DNA as a non-invasive biomarker: Accurate quantification using real time quantitative PCR without co-amplification of pseudogenes and dilution bias
Mitochondrial DNA as a non-invasive biomarker: Accurate quantification using real time quantitative PCR without co-amplification of pseudogenes and dilution bias
复制标题
DOI：
10.1016/j.bbrc.2011.06.067
复制
发表时间：
2011-08-19
影响因子：
3.1
通讯作者：
Cunningham, Phil
中科院分区：
文献类型：
--
作者：
Malik, Afshan N.;Shahni, Rojeen;Cunningham, Phil
循环线粒体DNA(MtDNA)是一种潜在的非侵入性细胞线粒体功能障碍的生物标志物，后者被认为是一系列人类疾病的核心。线粒体DNA的变化通常通过实时定量聚合酶链式反应测定线粒体DNA与核DNA的比值(mt/N)来确定。我们认为测量mt/N的方法需要改进，我们发现目前的方法至少存在以下三个问题中的一个：(1)由于线粒体基因组的大部分在核基因组中复制，许多常用的mtDNA引物共扩增核基因组中发现的同源假基因；(2)使用重复和/或高度可变的基因区域来进行核基因组的qPCR会导致错误；以及(3)当模板DNA稀释时，线粒体和核基因组的大小差异会导致“稀释偏差”。我们描述了一种基于PCR的方法，该方法利用人类线粒体基因组中没有在核基因组中复制的独特区域；核基因组中独特的单拷贝区域和模板处理来消除稀释偏差，以准确地定量人类样本中的mtDNA。(C)2011 Elsevier Inc.保留所有权利。
Circulating mitochondrial DNA (MtDNA) is a potential non-invasive biomarker of cellular mitochondrial dysfunction, the latter known to be central to a wide range of human diseases. Changes in MtDNA are usually determined by quantification of MtDNA relative to nuclear DNA (Mt/N) using real time quantitative PCR. We propose that the methodology for measuring Mt/N needs to be improved and we have identified that current methods have at least one of the following three problems: (1) As much of the mitochondrial genome is duplicated in the nuclear genome, many commonly used MtDNA primers co-amplify homologous pseudogenes found in the nuclear genome; (2) use of regions from genes such as beta-actin and 18S rRNA which are repetitive and/or highly variable for qPCR of the nuclear genome leads to errors; and (3) the size difference of mitochondrial and nuclear genomes cause a "dilution bias" when template DNA is diluted. We describe a PCR-based method using unique regions in the human mitochondrial genome not duplicated in the nuclear genome; unique single copy region in the nuclear genome and template treatment to remove dilution bias, to accurately quantify MtDNA from human samples. (C) 2011 Elsevier Inc. All rights reserved.