Mitochondria are involved in a number of diverse cellular functions, including energy production, metabolic regulation, apoptosis, calcium homeostasis, cell proliferation, and motility, as well as free radical generation. Mitochondrial DNA (mtDNA) is present at hundreds to thousands of copies per cell in a tissue-specific manner. mtDNA copy number also varies during aging and disease progression and therefore might be considered as a biomarker that mirrors alterations within the human body. Here, we present a new quantitative, highly sensitive droplet digital PCR (ddPCR) method, droplet digital mitochondrial DNA measurement (ddMDM), to measure mtDNA copy number not only from cell populations but also from single cells. Our developed assay can generate data in as little as 3 h, is optimized for 96-well plates, and also allows the direct use of cell lysates without the need for DNA purification or nuclear reference genes. We show that ddMDM is able to detect differences between samples whose mtDNA copy number was close enough as to be indistinguishable by other commonly used mtDNA quantitation methods. By utilizing ddMDM, we show quantitative changes in mtDNA content per cell across a wide variety of physiological contexts including cancer progression, cell cycle progression, human T cell activation, and human aging.

中文翻译：

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使用具有单细胞分辨率的液滴数字PCR定量测定线粒体DNA拷贝数。

线粒体参与许多不同的细胞功能，包括能量产生，代谢调节，细胞凋亡，钙稳态，细胞增殖和运动以及自由基的产生。线粒体DNA（mtDNA）以组织特异性方式存在于每个细胞中数百至数千个拷贝。mtDNA拷贝数在衰老和疾病发展过程中也会发生变化，因此可被视为反映人体内部变化的生物标记。在这里，我们提出了一种新的定量，高度灵敏的液滴数字PCR（ddPCR）方法，即液滴数字线粒体DNA测量（ddMDM），不仅可以测量细胞群体中的mtDNA拷贝数，还可以测量单个细胞中的mtDNA拷贝数。我们开发的分析方法可在短短3小时内生成数据，针对96孔板进行了优化，并且还允许直接使用细胞裂解液，而无需进行DNA纯化或核参考基因。我们表明，ddMDM能够检测mtDNA拷贝数足够接近以至于无法通过其他常用的mtDNA定量方法区分的样品之间的差异。通过利用ddMDM，我们显示了跨多种生理环境（包括癌症进展，细胞周期进展，人类T细胞活化和人类衰老）的每个细胞mtDNA含量的定量变化。