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Simultaneous Quantification of Mitochondrial DNA Copy Number and Deletion Ratio: A Multiplex Real-Time PCR Assay

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Simultaneous Quantification of Mitochondrial DNA Copy Number and Deletion Ratio: A Multiplex Real-Time PCR Assay

Nicole R Phillips et al. Sci Rep.

Abstract

Mitochondrial dysfunction is implicated in a vast array of diseases and conditions, such as Alzheimer's disease, cancer, and aging. Alterations in mitochondrial DNA (mtDNA) may provide insight into the processes that either initiate or propagate this dysfunction. Here, we describe a unique multiplex assay which simultaneously provides assessments of mtDNA copy number and the proportion of genomes with common large deletions by targeting two mitochondrial sites and one nuclear locus. This probe-based, single-tube multiplex provides high specificity while eliminating well-to-well variability that results from assaying nuclear and mitochondrial targets individually.

Figures

Figure 1
Figure 1. Location of mitochondrial targets.
The mtMajArc target is located within the major arc of the mtGenome (denoted by the dark arc between the origins of replication, OH and OL). It is spanned by 84% of the reported deletions, including the “common” deletion (denoted by the red arc between positions 8469-13447). The mtMinArc target is located in the D-loop of the mtGenome where no deletions have been reported.
Figure 2
Figure 2. Standard curve reproducibility.
Panel A- A representative real-time amplification plot of the standard DNA dilution series. The green horizontal line indicates the manual Cq threshold set at 0.04. Panel B- A representative standard curve regression. Note that the Y-intercept (provided in the table) is a function of the concentration of the standards; however, since the data are plotted by quantification standard number, the Y-intercepts are misrepresented (denoted by the broken x-axis). Panel C- Descriptive statistics for sixteen replicate standard curves analyzed on eight separate runs. Panel D- Descriptive statistics for the slope, amplification efficiency (Ex), Y-intercept (Y-int.), and correlation coefficient (R2) for eight runs with the standard curve analyzed in duplicate. All three targets are optimized, as indicated by the high R2 value (≥0.99) and high amplification efficiency (>90%). CI, confidence interval.
Figure 3
Figure 3. Reproducibility of the multiplex assay.
Absolute quantification was performed on eight samples (1–8) amplified in triplicate on three separate runs (nine replicates per sample) in order to assess the inter-assay variability. Panel A- The mean and standard deviation for each target are provided for each sample. Panel B- Descriptive statistics for mtDNA copy number of the nine replicates, calculated by the ratio of the mtMinArc target quantification result to the β2M quantification result. CI, confidence interval.
Figure 4
Figure 4. Assessment of major arc deletion detection resolution using a mixture series.
Two samples, one which harbors a major arc deletion at 61.5% heteroplasmy (mtDNADel) based on the multiplex quantification of the 1:0 neat sample, and one which does not harbor a known deletion (mtDNANormal), were mixed in varying proportions and analyzed in triplicate. The theoretical difference in mtDNA deletion ratio (mtDNADR) between each sample type is denoted with a triangle. Significant differences (α = 0.10) between the samples of varying deletion ratios are indicated with the brackets and asterisks; no significant difference was observed between the 1:0.75 mixture and the 1:1 mixture, the two mixtures which theoretically differ the least (mtDNADR difference of 4.4%). Therefore, this assay can detect differences in deletion ratio ≥ 5.9%.

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