title: Accurate measurement of mitochondrial DNA deletion level and copy number differences in human skeletal muscle.
abstract: 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.
authors.affiliation: Wellcome Trust Centre for Mitochondrial Research, Institute of Neuroscience, Newcastle University, Newcastle upon Tyne, United Kingdom.
authors.forename: John P
authors.initials: JP
authors.lastname: Grady
authors.affiliation: Wellcome Trust Centre for Mitochondrial Research, Institute of Neuroscience, Newcastle University, Newcastle upon Tyne, United Kingdom.
authors.forename: Julie L
authors.initials: JL
authors.lastname: Murphy
authors.affiliation: Wellcome Trust Centre for Mitochondrial Research, Institute of Neuroscience, Newcastle University, Newcastle upon Tyne, United Kingdom.
authors.forename: Emma L
authors.initials: EL
authors.lastname: Blakely
authors.affiliation: Department of Neurology, University of Texas Southwestern Medical Center and VA North Texas Medical Center, and Neuromuscular Center, Institute for Exercise and Environmental Medicine, Dallas, Texas, United States of America.
authors.forename: Ronald G
authors.initials: RG
authors.lastname: Haller
authors.affiliation: Wellcome Trust Centre for Mitochondrial Research, Institute of Neuroscience, Newcastle University, Newcastle upon Tyne, United Kingdom.
authors.forename: Robert W
authors.initials: RW
authors.lastname: Taylor
authors.affiliation: Wellcome Trust Centre for Mitochondrial Research, Institute of Neuroscience, Newcastle University, Newcastle upon Tyne, United Kingdom.
authors.forename: Doug M
authors.initials: DM
authors.lastname: Turnbull
authors.affiliation: Wellcome Trust Centre for Mitochondrial Research, Institute of Neuroscience, Newcastle University, Newcastle upon Tyne, United Kingdom.
authors.forename: Helen A L
authors.initials: HA
authors.lastname: Tuppen
mesh_terms: D000328:Adult; D056915:DNA Copy Number Variations; D004272:DNA, Mitochondrial / Q000235:genetics*; D005260:Female; D017353:Gene Deletion; D018628:Gene Dosage; D006801:Humans; D008297:Male; D008875:Middle Aged; D008931:Mitochondria, Muscle / Q000235:genetics*; D018482:Muscle, Skeletal / Q000502:physiology / Q000648:ultrastructure; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results
chemical_list: D004272:DNA, Mitochondrial