A Monochrome Multiplex Real-Time Quantitative PCR Assay for the Measurement of Mitochondrial DNA Content

摘要

Mitochondrial DNA copies per cell (mtDNA content) can fluctuate with cellular aging, oxidative stress, and mitochondrial dysfunction, and has been investigated in cancer, diabetes, HIV, and metabolic disease. mtDNA content testing in both clinical and basic settings is expected to increase as research uncovers its biological relevance. Herein, we present a novel mtDNA content assay developed on monochrome multiplex real-time quantitative PCR (MMqPCR) principles. This assay offers a greater than twofold improvement on time effectiveness and cost-effectiveness over conventional (monoplex) qPCR, as well as improved reproducibility given the reduced effects of human pipetting errors. The new MMqPCR method was compared with the gold standard monoplex qPCR assay on DNA from a variety of sources, including human whole blood, skeletal muscle, and commercial cell lines. The MMqPCR assay is reproducible (n?=?98,r?=?0.99,P? ?0.98,P? 82 pg of template DNA per reaction, with a minimum mtDNA/nuclear DNA ratio of 20, and is especially suitable for studies that require high throughput.