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

John P. Grady, Julie L. Murphy, Emma L. Blakely, Ronald G. Haller, Robert W. Taylor, Doug M. Turnbull, Helen A L Tuppen

Research output: Contribution to journalArticle

17 Citations (Scopus)

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.

Original languageEnglish (US)
Article numbere114462
JournalPLoS One
Volume9
Issue number12
DOIs
StatePublished - Dec 4 2014

Fingerprint

Mitochondrial DNA
Muscle
skeletal muscle
Skeletal Muscle
mitochondrial DNA
Assays
assays
Real-Time Polymerase Chain Reaction
quantitative polymerase chain reaction
Mitochondrial Diseases
DNA
mutation
therapeutics
Mutation
Molecules

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

Grady, J. P., Murphy, J. L., Blakely, E. L., Haller, R. G., Taylor, R. W., Turnbull, D. M., & Tuppen, H. A. L. (2014). Accurate measurement of mitochondrial DNA deletion level and copy number differences in human skeletal muscle. PLoS One, 9(12), [e114462]. https://doi.org/10.1371/journal.pone.0114462

Accurate measurement of mitochondrial DNA deletion level and copy number differences in human skeletal muscle. / Grady, John P.; Murphy, Julie L.; Blakely, Emma L.; Haller, Ronald G.; Taylor, Robert W.; Turnbull, Doug M.; Tuppen, Helen A L.

In: PLoS One, Vol. 9, No. 12, e114462, 04.12.2014.

Research output: Contribution to journalArticle

Grady, John P. ; Murphy, Julie L. ; Blakely, Emma L. ; Haller, Ronald G. ; Taylor, Robert W. ; Turnbull, Doug M. ; Tuppen, Helen A L. / Accurate measurement of mitochondrial DNA deletion level and copy number differences in human skeletal muscle. In: PLoS One. 2014 ; Vol. 9, No. 12.
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