Lookup NU author(s): Dr John Grady,
Dr Julie Murphy,
Professor Robert Taylor,
Professor Doug Turnbull,
Dr Helen Tuppen
This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).
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.
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
Online publication date: 04/12/2014
Acceptance date: 27/10/2014
ISSN (electronic): 1932-6203
Publisher: Public Library of Science
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