Quantitative mitochondrial DNA copy number determination using droplet digital PCR with single-cell resolution
Quantitative mitochondrial DNA copy number determination using droplet digital PCR with single-cell resolution
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DOI：
10.1101/gr.250480.119
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发表时间：
2019-11-01
期刊：
影响因子：
7
通讯作者：
Shay, Jerry W.
中科院分区：
文献类型：
--
作者：
O'Hara, Ryan;Tedone, Enzo;Shay, Jerry W.
线粒体参与许多不同的细胞功能，包括能量产生、代谢调节、凋亡、钙稳态、细胞增殖和运动以及自由基产生。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进展过程中也会发生变化，因此可能被认为是反映人体内变化的生物标志物。在这里，我们提出了一种新的定量，高灵敏度的液滴数字PCR（ddPCR）方法，液滴数字线粒体DNA测量（ddMDM），测量线粒体DNA拷贝数不仅从细胞群体，而且从单细胞。我们开发的检测方法可以在短短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物，而无需DNA纯化或核参考基因。我们表明，ddMDM是能够检测样本之间的差异，其mtDNA拷贝数是足够接近的其他常用的mtDNA定量方法无法区分。通过利用ddMDM，我们显示了在各种生理环境中每个细胞的mtDNA含量的定量变化，包括癌症进展，细胞周期进展，人类T细胞活化和人类衰老。
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.