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

Purpose: The mitochondrial genome (mtDNA) encodes thirteen essential proteins for oxidative phosphorylation. Depending on the cell type and stage of development, each cell contains an average of 10^3 to 10^4 copies of mtDNA. Current methods of mtDNA copy number quantification 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 components 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 ratio estimates. Methods: 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 (β2M). The accuracy of this assay was evaluated using a standard reference material (SRM2372a) and the precision was evaluated via replications. Results: This assay was optimized so both targets amplify with similar efficiency to give precise and accurate ratio estimates. The precision was demonstrated over repeated runs and shown to be accurate based on SRM quantification. Conclusion: This protocol demonstrates reproducible quantification in both mitochondrial and nuclear targets while reducing time and resources. The data generated can be used to indicate overall mitochondrial health and can be utilized in research areas such as aging, cancer, forensics and neurodevelopment.