Droplet Digital PCR Shows the D-Loop to be an Error Prone Locus for Mitochondrial DNA Copy Number Determination

Resource type
Date created
2018-07-30
Authors/Contributors
Author: Li, Brian
Author: Kim, BaRun
Author: Ching, Joyce
Abstract
Absolute quantification of mitochondrial DNA copy number (mCN) provides important insights in many fields of research including cancer, cardiovascular and reproductive health. Droplet digital PCR (ddPCR) natively reports absolute copy number, and we have developed a single-dye, multiplex assay to measure rat mCN that is accurate, precise and affordable. We demonstrate simple methods to optimize this assay and to determine nuclear reference pseudogene copy number to extend the range of mCN that can be measured with this assay. We evaluated two commonly used mitochondrial DNA reference loci to determine mCN, the ND1 gene and the D-Loop. Harnessing the absolute measures of ddPCR, we found that the D-Loop amplifies with a copy number of ~1.0–1.5 relative to other sites on the mitochondrial genome. This anomalous copy number varied significantly between rats and tissues (aorta, brain, heart, liver, soleus muscle). We advocate for avoiding the D-Loop as a mitochondrial reference in future studies of mCN. Further, we report a novel approach to quantifying immunolabelled mitochondrial DNA that provides single-cell estimates of mCN that closely agree with the population analyses by ddPCR. The combination of these assays represents a cost-effective and powerful suite of tools to study mCN.
Document
Published as
Scientific Reportsvolume 8, Article number: 11392 (2018). doi: 10.1038/s41598-018-29621-1
Publication title
Scientific Reports
Document title
Droplet Digital PCR Shows the D-Loop to be an Error Prone Locus for Mitochondrial DNA Copy Number Determination
Date
2018
Volume
8
Issue
11392
Publisher DOI
10.1038/s41598-018-29621-1
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Copyright is held by the author(s).
Scholarly level
Peer reviewed?
Yes
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s41598-018-29621-1.pdf 4.54 MB