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Accurate Measurement of Mitochondrial DNA Deletion Level and Copy Number Differences in Human Skeletal Muscle

Lookup NU author(s): Dr John Grady, Dr Julie Murphy, Professor Robert Taylor, Emeritus Professor Doug Turnbull, Dr Helen Tuppen

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This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).

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.

Publication metadata

Author(s): Grady JP, Murphy JL, Blakely EL, Haller RG, Taylor RW, Turnbull DM, Tuppen HAL

Publication type: Article

Publication status: Published

Journal: PLoS One

Year: 2014

Volume: 9

Issue: 12

Online publication date: 04/12/2014

Acceptance date: 27/10/2014

Date deposited: 19/02/2015

ISSN (electronic): 1932-6203

Publisher: Public Library of Science

URL: http://dx.doi.org/10.1371/journal.pone.0114462

DOI: 10.1371/journal.pone.0114462

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