Optimization and Evaluation of qPCR Duplex Assay for mtDNA Copy Number Quantification

Purpose: The mitochondrial genome (mtDNA) encodes thirteen essential proteins in oxidative phosphorylation, the cell's primary energy-generating process. Depending on the cell type and stage of development, each cell contains an average of 103 to 104 copies of mtDNA. Current methods of quantification of mtDNA copy number can be imprecise due to low efficiencies of assays and inherent imbalance of mtDNA copy number with nuclear DNA (nDNA) copy number. Accurate quantification of both mtDNA and nDNA is important when calculating the ratio of mtDNA to nDNA. The goal of this project is to optimize a duplex assay that will give precise and accurate estimates in human samples. Methods: Here we employ synthetic oligomer standards for an absolute real-time qPCR assay. The significance of using absolute qPCR is that the standard curve allows for the direct comparison of unknowns to obtain a copy number. The mitochondrial target is a site in the minor arc (MinArc), and the nuclear target is a single copy locus ([beta]2M). The accuracy of this assay was evaluated using a standard reference material (SRM2372a) and the precision was evaluated via replications. Results: This design resulted in high R2 values for the standards as well as sufficiently high efficiencies. The precision of the assay was analyzed over 6 replicated runs and was deemed effectively reproducible. The accuracy was assessed with the use of a standard reference material (SRM 2372a) and was found to be problematic [Romsos et al., 2018]. This could be from a possible dilution bias of the SRM, effectively changing the copy number ratios in a difficult to predict way [Malik et al., 2011]. An attempt to mathematically correct the data was made but did not provide any solution. Conclusion: The optimization of this assay is ongoing due to the error in accuracy. The assay has proven to be precise and reproducible with sufficient efficiency. Possible future directions include sonication of samples and SRMs to examine if dilution bias could be the cause of inaccurate SRM quantification. Other methods of possibly reducing dilution bias mentioned in Malik et al. [2011] include manual shearing and the use of DNA carriers such as tRNA. Another avenue of future research could include a different method of mathematically correcting the data post run to improve accuracy. This assay has the potential to provide data which can be used to indicate overall mitochondrial health and can be utilized in various research areas such as aging, cancer, forensics and neurodevelopment.