Journal
PLOS ONE
Volume 9, Issue 12, Pages -Publisher
PUBLIC LIBRARY SCIENCE
DOI: 10.1371/journal.pone.0114462
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Funding
- Wellcome Trust Centre for Mitochondrial Research [906919]
- National Institutes of Health (National Institute of Arthritis, Musculoskeletal and Skin Diseases, NIAMS) [RO1-AR050597]
- MRC [G0700718, MR/K006312/1, MR/K000608/1] Funding Source: UKRI
- Medical Research Council [MR/K006312/1, G0700718, MR/K000608/1] Funding Source: researchfish
- National Institute for Health Research [NF-SI-0510-10187] Funding Source: researchfish
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Accurate and reliable quantification of the abundance of mitochondrial DNA (mtDNA) molecules, both wild-type and those harbouring pathogenic mutations, is important not only for understanding the progression of mtDNA disease but also for evaluating novel therapeutic approaches. A clear understanding of the sensitivity of mtDNA measurement assays under different experimental conditions is therefore critical, however it is routinely lacking for most published mtDNA quantification assays. Here, we comprehensively assess the variability of two quantitative Taqman real-time PCR assays, a widely-applied MT-ND1/MT-ND4 multiplex mtDNA deletion assay and a recently developed MT-ND1/B2M singleplex mtDNA copy number assay, across a range of DNA concentrations and mtDNA deletion/copy number levels. Uniquely, we provide a specific guide detailing necessary numbers of sample and real-time PCR plate replicates for accurately and consistently determining a given difference in mtDNA deletion levels and copy number in homogenate skeletal muscle DNA.
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