Methodological influences on circulating cell-free-mitochondrial and nuclear DNA concentrations in response to chronic stress

Abstract

Background Mitochondria are versatile eukaryotic organelles that play a crucial role in the body’s stress response. Prolonged stress exposure can cause structural and functional alterations, leading to mitochondrial DNA (mtDNA) damage and subsequent release of mtDNA into the circulation. Cell-free circulating mtDNA (ccf-mtDNA) is a potential biomarker indicating cellular damage and stress. In this study we investigated the applicability of ccf-mtDNA and cf-nDNA as biomarkers of chronic stress in healthy subjects. Methods and results We developed a quantitative polymerase chain reaction (qPCR) assay to directly measure ccf-mtDNA in human blood plasma samples, addressing numerous challenges specifically related to ccf-mtDNA quantification. We validated our 68 bp target assay based on the FDA, International Organization for Standardization (ISO) and Clinical & Laboratory Standards Institute (CLSI) guidelines for assay development, including parameters such as limit of blank (LOB), limit of detection (LOD) and limit of quantification (LOQ). Furthermore, we implemented incurred samples analysis and inter-plate samples to ensure reliability and reproducibility of the assay. In addition, we evaluated the effects of centrifugation forces on ccf-mtDNA and cf-nDNA concentrations in native plasma samples and showed that mainly ccf-mtDNA is strongly affected by centrifugation forces. We found a significant negative correlation between ccf-mtDNA levels and chronic stress. In contrast, cf-nDNA levels were not affected in response to chronic stress. Conclusion ccf-mtDNA can directly and reliably quantified in unpurified plasma samples. However, the ccf-mtDNA levels in plasma samples of healthy subjects are close the LOQ, showing that the assay is not yet suitable for all conditions.