Fazzini 2016 Abstract Mito Xmas Meeting Innsbruck
|Determination of mitochondrial copy number as versatile tool in epidemiology.|
Alterations of mitochondrial DNA (mtDNA) copy number appear to be associated with several pathologies including encephalopathies and neuropathies as well as the process of aging [1-2].
The aim of this study was to set up a reliable quantitative PCR based assay for mitochondrial DNA copy number determination meeting quality requirements for mtDNA specificity.
We established a duplex quantitative PCR assay that allows for targeting a single copy nuclear gene (ß2-microglobulin) and the mtDNA (t-RNA Leu) simultaneously.
The use of a plasmid containing both targets in a 1:1 ratio was used to normalize against differences in emission intensities of the fluorescent dyes VIC and FAM.
QPCR on the serial dilution of the calibrator plasmid revealed that the FAM dye emission signal exceeded the VIC signal, resulting in a ΔCT value of up to 1.2 cycles corresponding to more than a double amount of molecules. Using the plasmid calibrator with internal positive controls reduced the intra-assay variability from 21% (uncorrected) to 7% (plasmid corrected). We evaluated the applicability of the method by using DNA samples that were isolated with different methods and revealed significantly different numbers of mtDNA copies (copy number ratio: salting out/magnetic beads = 1.65).
We developed a sensitive and robust assay for mitochondrial copy number detection relative to nuclear DNA. The use of the dual insert calibrator plasmid allows for correction against unequal emission intensities of the differently fluorescence labelled targets. Furthermore, we discovered that the diverse extraction methods selectively isolate different DNA molecules within a sample.
Labels: MiParea: mtDNA;mt-genetics, nDNA;cell genetics
Event: B1, Oral
- Fazzini F(1), Hicks AA(2), Kronenberg F(1), Fendt L(1)
- Div Genetic Epidemiology, Medical Univ Innsbruck, Austria
- Center Biomedicine, European Acad Bolzano/Bozen (EURAC), Italy
- Tuomi JM, Voorbraak F, Jones DL, Ruijter JM (2010) Bias in the Cq value observed with hydrolysis probe based quantitative PCR can be corrected with the estimated PCR efficiency value. Methods 50:313–22
- Reznik E, Miller ML, Şenbabaoğlu Y, Riaz N, Sarungbam J, Tickoo SK, Al-Ahmadie HA, Lee W, Seshan VE, Hakimi AA, Sander C (2016) Mitochondrial DNA copy number variation across human cancers. eLife 5:1–20.