Accurate Measurement of Cellular and Cell-Free Circulating Mitochondrial DNA Content from Human Blood Samples Using Real-Time Quantitative PCR

Hannah Rosa, Afshan N. Malik*

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

10 Citations (Scopus)

Abstract

Changes in circulating mitochondrial DNA (mtDNA) are widely used to indicate mitochondrial dysfunction in common non-genetic diseases where mitochondrial dysfunction may play a role. However, the methodology being used is not always specific and reproducible, and most studies use whole blood rather than evaluating cellular and cell-free mtDNA separately. Cellular mtDNA is contained within the mitochondrion and encodes vital subunits of the OXPHOS machinery. Conversely, cell-free mtDNA can have harmful effects, triggering inflammatory responses and potentially contributing to pathogenic processes. In this chapter, we describe a protocol to accurately measure the amount of cellular and cell-free human mtDNA in peripheral blood. Absolute quantification is carried out using real-time quantitative PCR (qPCR) to quantify cellular mtDNA, measured as the mitochondrial genome to nuclear genome ratio (designated the Mt/N ratio) in whole blood and peripheral blood mononuclear cells (PBMCs) and the number of mtDNA copies per μL in plasma and serum. We describe how to (1) separate whole blood into PBMCs, plasma, and serum fractions, (2) prepare DNA from each of these fractions, (3) prepare dilution standards for absolute quantification, (4) carry out qPCR for either relative or absolute quantification from test samples, (5) analyze qPCR data, and (6) calculate the sample size to adequately power studies. The protocol presented here is suitable for high-throughput use and can be modified to quantify mtDNA from other body fluids, human cells, and tissues.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc
Pages247-268
Number of pages22
DOIs
Publication statusPublished - 2021

Publication series

NameMethods in Molecular Biology
Volume2277
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • Absolute quantification
  • Circulating mtDNA
  • Mitochondrial DNA
  • Mt/N ratio
  • mtDNA
  • mtDNA content
  • mtDNA copy number
  • PBMCs
  • Plasma
  • qPCR
  • Serum

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