Absolute mtDNA Copy Number Analysis
The TDMM Core offers absolute quantitation of mitochondrial genome copy numbers. Mitochondria contain multiple circular mitochondrial genomes (mtDNA) which encode proteins for oxidative phosphorylation and ribosomal RNAs for protein translation. The loss of mtDNA integrity, both reduced copy number and increased mutations, is implicated in diseases including diabetes, cancer, and sarcopenia, and disrupted mitochondrial function and loss of tissue homeostasis. Therefore, methods to quantify mtDNA copies are important for characterization of clinical samples and disease models. Existing quantitative PCR (qPCR) methods to quantify mtDNA copy number provide only relative quantitation and are problematic to normalize to different template input amounts and across tissues/sample types. As well, existing methods cannot quantify mtDNA copy number in subcellular isolates, such as isolated mitochondria and neuronal synaptic terminals, which lack nuclear genomic DNA. We have created a novel absolute mtDNA copy number quantitation method for human, rat, and mouse samples. This approach uses chip-based digital polymerase chain reaction (dPCR) and fluorogenic assays to count mtDNA copies and is applicable to subcellular isolates and whole cell preparations. For whole cell samples mtDNA counts can be normalized to nuclear genome copy number to provide a measure of mtDNA per cell. This simple and cost effective dPCR approach demonstrates precise and accurate mtDNA copy number quantitation.
To perform mtDNA copy number analysis ~100ng of genomic DNA is required per sample. Core users should isolate gDNA by standard methods and quantify by spectrophotometry. Currently human, mouse, and rat samples can be analyzed. Two versions of the analysis are available: 1) mtDNA absolute quantitation and 2) absolute quantitation normalized to the number of nuclear genomes/cells.
Absolute mtDNA quantitation dPCR workflow. A) Total genomic DNA is isolated from tissue/cells and mixed with proper assay components. The reactions are distributed across a chip with 20,000 856 pl wells. Template is diluted to a point where there is either 0 or 1 copies per well, i.e., digital. Reactions are then cycled to end-point and fluorescence is read in each well. Those wells that were loaded with template are positive wells, while those wells that were filled without template are negative. Based on the count of fluorescent positive and negative wells and using a Poisson distribution, the number of target copies can be calculated per microliter. B) Representative raw fluorescence data from a mtDNA assay (left) and a nuclear DNA reference assay (right) showing chips with positive and negative wells (top) and corresponding well counts of fluorescent intensities showing a bimodal distribution of positive and negative wells.
Total sample DNA quantification is carried out using fluorescent-based PicoGreen assay (Life Technologies). Digital PCR is then performed according to manufacturer’s instructions by mixing 3.33 µl diluted template DNA (concentrations of DNA are first determined by performing test dilution series) with 16.5 µl Quantstudio 3D master mix, 3.33 µl TaqMan assay, and 9 µl water (32.16 µl [enough for two chips with excess]) (Life Technologies). Reactions are loaded onto Quantstudio 3D digital PCR chips using the Quantstudio 3D chip loader according to manufacturer’s instructions (Life Technologies). Chips are then sealed and cycled on a GeneAmp PCR system 9700 with a flatblock attachment using the following conditions; Stage 1, 96° C for 10 minutes, Stage 2, 60° C for 2 minutes then 98° C for 30 seconds, repeat Stage 2 39 times, Stage 3, 60° C for 2 minutes and an infinite 10° C hold. Chips are then read in the Quantstudio 3D chip reader to obtain raw fluorescent values (Life Technologies). Quality check of the chips and counting of positive and negative wells in order to determine copies/microliter are carried out on the Quantstudio 3D AnalysisSuite cloud software (https://apps.lifetechnologies.com/quantstudio3d/index.html; LifeTechnologies) using the Absolute Quantification module. Data can then be shared with other investigators through the cloud application or can be exported and sent in conventional spreadsheet format.
Total genomic DNA (both nuclear and mitochondrial) should be extracted from tissues using a standard column preparation (e.g., Qiagen AllPrep DNA/RNA) or using solution (Trizol/TriReagent) methods. Core users should prepare their own DNA according their experience as isolation techniques vary according to sample type (tissues, cells, blood). DNA quality should be determined by spectrophotometry with attention to organic contamination (absorbance at 230nm) as high levels of organic contamination can inhibit dPCR reactions. 1ug of DNA is required for this analysis.