A single-tube multiplex qPCR assay for mitochondrial DNA (mtDNA) copy number assessment

Abstract

Abstract Objective Detection of mtDNA copy number is required for diagnosis of mtDNA depletion. Multiplex quantification of mtDNA in blood samples was claimed via normalizing to a nuclear single copy gene using qPCR. This is not possible in high mtDNA samples due to template abundance. Multiplex qPCR assays cannot be normalized to single copy sequences of the nuclear genome. Methods mtDNA quantification was tested normalizing to a single copy nuclear gene via singleplex and multiplex reactions. Failure in normalization directed to design and test targeting multi-copy 18S rDNA gene with success. mtDNA quantification was standardized both in separate and multiplexed single-tube reactions based on molecular beacon technology. Results mtDNA copy number assessment cannot be normalized to a single copy sequence in high-copy-number tissues. However, normalizing mtDNA to the nuclear 18S rDNA multiple copy sequence is amenable to be standardized in single tube. When compared, multiplexing exhibited higher resolution power for quantification of mtDNA in various samples from the most abundant to the scant ones. Conclusion We describe a multiplex assay that can be translated as a standard technique for single-tube quantification of mtDNA copy number. Our findings show higher accuracy and reproducibility over canonical approach, reducing cost and error rate.