Development of a real-time PCR assay for the direct quantification of circulating cell-free mitochondrial DNA in human blood plasma: preanalytical considerations, challenges, and clinical implications
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Abstract
Mitochondria are multifunctional organelles increasingly recognized as key players in stress research
due to their ability to sense and respond to external and internal stressors. As such, they are central
to our understanding of cellular resilience and signaling pathways in response to acute or chronic
stress. One consequence of prolonged mitochondrial stress and subsequent dysfunction is the release
of mitochondrial components, such as mitochondrial DNA (mtDNA) into the circulation. In this
context, circulating cell free nucleic acids (cf-DNA) have emerged as biomarker in both pathological
and physiological conditions. To date the relevance of cf-DNA, particularly in liquid biopsy, is well
established and widely applied in clinical routine and medical practice. Quantifying circulating cell
free mtDNA (ccf-mtDNA) therefore offers a valuable tool for gaining insights into mitochondrial
responses and the broader cellular mechanisms activated under stressful conditions. However,
quantifying ccf-mtDNA remains challenging due to several technical and pre-analytical
considerations. A method for measuring and quantifying ccf-mtDNA in human blood plasma
samples using direct quantitative polymerase chain reaction (qPCR) without the need for prior
isolation is presented in this thesis. This approach shows significant advantages compared to
traditional methods including reduced sample processing time, lower risk of sample loss, cost
efficiency and improved overall assay performance. The establishment of the qPCR-assay
implemented serval recommendation guidelines to ensure accuracy and reproducibility. To evaluate
the applicability of the newly developed assay in clinical samples, ccf-mtDNA concentrations were
measured in healthy individuals in respect to their chronic stress load. The results revealed a negative
correlation between ccf-mtDNA and the perceived chronic stress in healthy individuals, whereas no
such correlation was observed for cf-nDNA. To further investigate this stress-associated relationship,
ccf-mtDNA kinetics was assessed after acute stress induction using the Trier Social Stress Test
(TSST), a well-established and controlled procedure for eliciting physiological stress response in both
healthy individuals and panic disorder (PD) patients. The findings of the present thesis demonstrated
that while ccf-mtDNA concentrations remained unchanged in the healthy control group, they
showed a significant decrease 10- minutes after acute stress induction in the PD patients. Taken
together the results provide a solid foundation for the direct quantification of ccf-mtDNA
concentrations in clinical samples and open new opportunities to further investigate its role in stress-
research and a wide range of conditions such as inflammatory diseases, cancer, neurodegenerative
disorders, and more.