Measuring Single-Cell Mitochondrial DNA Copy Number and Heteroplasmy Using Digital Droplet Polymerase Chain Reaction

Here we present a protocol for measuring absolute mitochondrial (mt)DNA copy number and mtDNA deletion heteroplasmy levels in single cells.

The mammalian mitochondrial (mt)DNA is a small, circular, double-stranded, intra-mitochondrial DNA molecule, encoding 13 subunits of the electron transport chain. Unlike the diploid nuclear genome, most cells contain many more copies of mtDNA, ranging from less than 100 to over 200,000 copies depending on cell type. MtDNA copy number is increasingly used as a biomarker for a number of age-related degenerative conditions and diseases, and thus, accurate measurement of the mtDNA copy number is becoming a key tool in both research and diagnostic settings. Mutations in the mtDNA, often occurring as single nucleotide polymorphisms (SNPs) or deletions, can either exist in all copies of the mtDNA within the cell (termed homoplasmy) or as a mixture of mutated and WT mtDNA copies (termed heteroplasmy). Heteroplasmic mtDNA mutations are a major cause of clinical mitochondrial pathology, either in rare diseases or in a growing number of common late-onset diseases such as Parkinson's disease. Determining the level of heteroplasmy present in cells is a critical step in the diagnosis of rare mitochondrial diseases and in research aimed at understanding common late-onset disorders where mitochondria may play a role. MtDNA copy number and heteroplasmy have traditionally been measured by quantitative (q)PCR-based assays or deep sequencing. However, the recent introduction of ddPCR technology has provided an alternative method for measuring both parameters. It offers several advantages over existing methods, including the ability to measure absolute mtDNA copy number and sufficient sensitivity to make accurate measurements from single cells even at low copy numbers. Presented here is a detailed protocol describing the measurement of mtDNA copy number in single cells using ddPCR, referred to as droplet generation PCR henceforth, with the option for simultaneous measurement of heteroplasmy in cells with mtDNA deletions. The possibility of expanding this method to measure heteroplasmy in cells with mtDNA SNPs is also discussed.

Mammalian mitochondrial (mt)DNA is a small (approx. 16.5 Kb), circular DNA genome residing in the mitochondrial matrix that encodes 37 genes, comprising two rRNAs, 22 tRNAs, and 13 protein-coding genes¹. Unlike the nuclear genome, which contains one (haploid) or two (diploid) copies of each gene per cell, mtDNA is present in multiple copies in the mitochondria of each cell, ranging from tens of copies (e.g., mature spermatocytes) to hundreds of thousands of copies (e.g., oocytes)^2,3. A consequence of this multi-copy nature is that mutations in the mtDNA genome, which may exist as single....

All experiments followed the ARRIVE guidelines and were approved by the University of Cambridge Animal Welfare Ethical Review Body (AWERB).

NOTE: All sample preparation steps prior to droplet generation must be performed in a clean pre-PCR work area, ideally in a UV-sterilized cabinet where possible. The protocol described here uses specific droplet generation PCR equipment (see Table of Materials), and while the general method should be applicable to other systems, it is reco.......

Following droplet generation, a clear layer of opaque droplets is visible floating on top of the oil phase in each well (Figure 1B). Droplet formation can be adversely affected by the presence of detergents in the input lysate when performing experiments on single cells. Using the lysis protocol described in 2.1.2., droplet yields above the recommended level of 10,000 are routinely achieved, despite the presence of a small residual amount of TWEEN-20 in the final sample (

The protocol described here is applicable across a wide range of cell types and species in addition to those discussed above, although careful optimization of new assay designs will be key to ensure that accuracy and repeatability of the method are maintained when moving away from previously validated primer/probe combinations. When working with single cells it is vital to ensure that sample collection is performed as accurately as possible (e.g., using stringent single-cell parameters when sorting cells by FACS) to ensu.......

Thank you to Dr. L Bozhilova for advice on the statistical analysis of droplet generation PCR data. Thank you to Dr. H Zhang for providing the oocytes used to generate data in Figure 3C and Figure 4B. This work was carried out by SPB at the Medical Research Council Mitochondrial Biology Unit (MC_UU_00015/9), University of Cambridge, and funded by a Wellcome Trust Principal Research Fellowship held by PFC (212219/Z/18/Z).