Human mitochondrial DNA (mtDNA) is a small maternally inherited DNA, present in hundreds of copies in a single human cell. Thus, despite its small size, the mitochondrial genome plays a crucial role in the metabolic homeostasis of the cell. Alterations of mitochondrial function, dynamics, and biogenesis have been observed in various metabolic disorders, including aging, cancer, diabetes, and obesity.
The aims of this project are: first to investigate if there is a variation in mtDNA copy number in the general population and second to investigate through GWAS whether nuclear variants can be associated with a relative change in mtDNA copy numbers per cell. Furthermore we interested whether a variation in mtDNA copy number can be related to a particular pathogenic phenotype. We plan to analyze 5000 DNA samples from peripheral blood derived from CHRIS, a prospective epidemiological research study conducted in South Tyrol (EURAC-Bolzano).
We established a duplex quantitative PCR assay that allows for targeting a single copy nuclear gene and the mtDNA, simultaneously. The use of a plasmid containing both targets in a 1:1 ratio was used to normalize against differences in emission intensities of the fluorescent dyes. For our validation test we run a positive control in different experiments. We obtained a lower intra-assay variability when using our plasmid correction model as compare to the uncorrected row data. This proves that the plasmid correction method improves the accuracy of the analysis.
We set up a universal and precise method to determine the number of mitochondrial copy per diploid cell. The use of the dual insert calibrator plasmid allows the correction against the misestimation of the mtDNA copy number resulting from unequal emission intensities of the differently fluorescence labeled targets. Furthermore, our calculation method takes into account the real PCR efficiency during the exponential phase of the amplification reaction.
