A Monochrome Multiplex Real-Time Quantitative PCR Assay for the Measurement of Mitochondrial DNA Content
A Monochrome Multiplex Real-Time Quantitative PCR Assay for the Measurement of Mitochondrial DNA Content
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DOI：
10.1016/j.jmoldx.2018.05.001
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发表时间：
2018-09-01
影响因子：
4.1
通讯作者：
Cote, Helene C. F.
中科院分区：
文献类型：
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作者：
Hsieh, Anthony Y. Y.;Budd, Matthew;Cote, Helene C. F.
每个细胞的线粒体DNA拷贝数（mtDNA含量）可以随着细胞衰老、氧化应激和线粒体功能障碍而波动，并且已经在癌症、糖尿病、HIV和代谢疾病中进行了研究。随着研究揭示其生物学相关性，临床和基础环境中的mtDNA含量检测预计将增加。在此，我们提出了一种新的线粒体DNA含量检测单色多重实时定量PCR（MMqPCR）的原则。与常规（单重）qPCR相比，该测定在时间有效性和成本有效性方面提供了两倍以上的改进，并且鉴于人类移液错误的影响降低，重现性得到了改善。新的MMqPCR方法与金标准单重qPCR检测方法在来自各种来源的DNA上进行了比较，包括人全血、骨骼肌和商业细胞系。MMqPCR测定是可重复的（n = 98，r = 0.99，P < 0.0001），并且与单重qPCR测定高度相关（n = 160，r > 0.98，P < 0.0001）。由多名操作员独立确定的试验内和试验间变异性范围分别为4.3%-7.9%和2.9%-9.2%。这种稳健的检测方法每次反应可以定量>82 pg的模板DNA，最小mtDNA/核DNA比为20，特别适用于需要高通量的研究。
Mitochondrial DNA copies per cell (mtDNA content) can fluctuate with cellular aging, oxidative stress, and mitochondrial dysfunction, and has been investigated in cancer, diabetes, HIV, and metabolic disease. mtDNA content testing in both clinical and basic settings is expected to increase as research uncovers its biological relevance. Herein, we present a novel mtDNA content assay developed on monochrome multiplex real-time quantitative PCR (MMqPCR) principles. This assay offers a greater than twofold improvement on time effectiveness and cost-effectiveness over conventional (monoplex) qPCR, as well as improved reproducibility given the reduced effects of human pipetting errors. The new MMqPCR method was compared with the gold standard monoplex qPCR assay on DNA from a variety of sources, including human whole blood, skeletal muscle, and commercial cell lines. The MMqPCR assay is reproducible (n = 98, r = 0.99, P < 0.0001) and highly correlated to the monoplex qPCR assay (n = 160, r > 0.98, P < 0.0001). Intra-assay and interassay variabilities, as established independently by multiple operators, range between 4.3% and 7.9% and between 2.9% and 9.2%, respectively. This robust assay can quantify >82 pg of template DNA per reaction, with a minimum mtDNA/nuclear DNA ratio of 20, and is especially suitable for studies that require high throughput.