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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.

SUBMITTER: Grady JP 

PROVIDER: S-EPMC4256439 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Accurate measurement of mitochondrial DNA deletion level and copy number differences in human skeletal muscle.

Grady John P JP   Murphy Julie L JL   Blakely Emma L EL   Haller Ronald G RG   Taylor Robert W RW   Turnbull Doug M DM   Tuppen Helen A L HA  

PloS one 20141204 12


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  ...[more]

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