Circulating Mitochondrial DNA: Quantitative Analysis

Abstract

Previous studies have demonstrated the existence of circulating mitochondrial DNA in plasma and serum. However, the quantitative parameters and physical characteristics of circulating mitochondrial DNA are unknown. A mitochondrial-sequence-specific real-time quantitative PCR assay was developed. The specificity of the assay for mitochondrial DNA detection was evaluated with a cell line (rho0) devoid of mitochondrion. Experiments were conducted in three modules that were designed to evaluate the following, respectively: (1) the effects of different blood processing protocols on the quantitative analysis of circulating mitochondrial DNA; (2) the existence of particle-associated and free forms of mitochondrial DNA in plasma through filtration and ultracentrifugation experiments; and (3) the size characteristics of the particles associated with circulating mitochondrial DNA, by filtering plasma through filters of different pore sizes. The results of module 1 revealed that the concentrations of circulating mitochondrial DNA in differentially-processed plasma aliquots were significantly different. Data from module 2 further demonstrated that a significant fraction of mitochondrial DNA in plasma was filterable or pelletable upon ultracentrifugation. Module 3 demonstrated that filters of different pore sizes could remove mitochondrial DNA from plasma to varying extents. The data of these experiments have thus demonstrated that both particle-associated and free mitochondrial DNA molecules are present in plasma and their respective concentrations are significantly affected by how plasma is harvested from whole blood. The heterogeneous nature of circulating mitochondrial DNA may need to be taken into account in the design of future studies.