Quantitative analysis of circulating mitochondrial DNA in plasma
Quantitative analysis of circulating mitochondrial DNA in plasma
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DOI：
10.1373/49.5.719
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发表时间：
2003-05-01
影响因子：
9.3
通讯作者：
Lo, YMD
中科院分区：
文献类型：
--
作者：
Chiu, RWK;Chan, LYS;Lo, YMD
背景：最近的研究表明血浆和血清中存在循环线粒体DNA，但循环线粒体DNA的浓度和物理特性尚不清楚。本研究的目的是开发一种定量测定健康人血浆中线粒体DNA的方法。方法：我们采用实时定量PCR方法，并评估该方法在线粒体缺失细胞系（rho(o)）中检测线粒体DNA的特异性。通过三个模块的实验，研究了循环线粒体DNA的浓度和物理特性。在模块1中，我们评估了来自四种血液处理方案的血浆等分液中线粒体DNA的浓度。在模块2中，我们通过对血浆进行过滤和超离心，研究了血浆中线粒体DNA的颗粒相关形式和自由形式的存在。在模块3中，我们使用不同孔径的过滤器来研究循环线粒体DNA颗粒相关部分的大小特征。结果：线粒体DNA特异性实时定量PCR具有5个数量级的动态范围和灵敏度，可以检测血浆中线粒体DNA的一个拷贝。在模块1中，我们发现不同方法处理的血浆等分液中循环线粒体DNA的数量存在显著差异。模块2的数据显示，血浆中有很大一部分线粒体DNA可以通过超离心过滤或成球。模块3演示了不同孔径的过滤器对血浆中线粒体DNA的去除程度不同。结论：血浆中存在颗粒相关DNA和游离线粒体DNA，它们各自的浓度受到从全血中采集血浆的过程的影响。这些结果可能对未来循环线粒体DNA研究的设计有启示意义。在不同的疾病条件下测量。(C) 2003年美国临床化学学会。
Background: Recent studies have demonstrated the existence of circulating mitochondrial DNA in plasma and serum, but the concentrations and physical characteristics of circulating mitochondrial DNA are unknown. The aim of this study was to develop an assay to quantify mitochondrial DNA in the plasma of healthy individuals.Methods: We adopted a real-time quantitative PCR approach and evaluated the specificity of the assay for detecting mitochondrial DNA with a cell line (rho(o)) devoid of mitochondria. The concentrations and physical characteristics of circulating mitochondrial DNA were investigated by experiments conducted in three modules. In module 1, we evaluated the concentrations of mitochondrial DNA in plasma aliquots derived from four blood-processing protocols. In module 2, we investigated the existence of both particle-associated and free forms of mitochondrial DNA in plasma by subjecting plasma to filtration and ultracentrifugation. In module 3, we used filters with different pore sizes to investigate the size characteristics of the particle-associated fraction of circulating mitochondrial DNA.Results: The mitochondrial DNA-specific, real-time quantitative PCR had a dynamic range of five orders of magnitude and a sensitivity that enabled detection of one copy of mitochondrial DNA in plasma. In module 1, we found significant differences in the amounts of circulating mitochondrial DNA among plasma aliquots processed by different methods. Data from module 2 revealed that a significant fraction of mitochondrial DNA in plasma was filterable or pelletable by ultracentrifugation. Module 3 demonstrated that filters with different pore sizes removed mitochondrial DNA from plasma to different degrees.Conclusions: Both particle-associated and free mitochondrial DNA are present in plasma, and their respective concentrations are affected by the process used to harvest plasma from whole blood. These results may have implications in the design of future studies on circulating mitochondrial DNA. measured in different disease conditions. (C) 2003 American Association for Clinical Chemistry.