Plasmid-normalized quantification of relative mitochondrial DNA copy number.
Federica FazziniBernd SchöpfMichael BlatzerStefan CoassinAndrew A HicksFlorian KronenbergLiane FendtPublished in: Scientific reports (2018)
Alterations of mitochondrial DNA (mtDNA) copy number have been associated with a wide variety of phenotypes and diseases. Unfortunately, the literature provides scarce methodical information about duplex targeting of nuclear and mtDNA that meets the quality criteria for qPCR. Therefore, we established a method for mtDNA copy number quantification using a quantitative PCR assay that allows for simultaneous targeting of a single copy nuclear gene (beta-2-microglobulin) and the t-RNALeu gene on the mtDNA. We include a plasmid containing both targets in order to normalize against differences in emission intensities of the fluorescent dyes Yakima Yellow and FAM. Applying the plasmid calibrator on an internal control reduced the intra-assay variability from 21% (uncorrected) to 7% (plasmid-corrected). Moreover, we noted that DNA samples isolated with different methods revealed different numbers of mtDNA copies, thus highlighting an important influence of the pre-analytical procedures. In summary, we developed a precise assay for mitochondrial copy number detection relative to nuclear DNA. Our method is applicable to comparative mitochondrial DNA copy number studies since the use of the dual insert plasmid allows correcting for the unequal emission intensities of the different fluorescent labels of the two targets.
Keyphrases
- copy number
- mitochondrial dna
- escherichia coli
- crispr cas
- genome wide
- high throughput
- dna methylation
- circulating tumor
- single molecule
- systematic review
- quantum dots
- living cells
- label free
- cell free
- cancer therapy
- oxidative stress
- high resolution
- gene expression
- single cell
- social media
- quality improvement
- nucleic acid
- transcription factor
- loop mediated isothermal amplification