Edit Abstract: Fazzini 2016 Abstract Mito Xmas Meeting Innsbruck

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Abstract:	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.
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MiP areas:	Gender Respiration Instruments;methods mt-Biogenesis;mt-density mt-Structure;fission;fusion mt-Membrane mtDNA;mt-genetics nDNA;cell genetics Genetic knockout;overexpression Comparative MiP;environmental MiP Developmental biology Exercise physiology;nutrition;life style mt-Medicine Patients Pharmacology;toxicology mt-Awareness
Pathological states:	COPD Aging;senescence Alzheimer's Autism Cancer Cardiovascular Diabetes Inherited Infectious Myopathy Neurodegenerative Obesity Parkinson's Sepsis Other
Stress conditions:	Hypoxia Cell death Cryopreservation Ischemia-reperfusion Permeability transition Oxidative stress;RONS Temperature Mitochondrial disease
Organism:	Pig Human Mouse Rat Guinea pig Bovines Horse Dog Rabbit Cat Other mammals Chicken Zebrafish Drosophila Artemia Caenorhabditis elegans Saccharomyces cerevisiae Birds Reptiles Amphibians Fishes Chelicerates Crustaceans Hexapods Myriapods Annelids Molluscs Nematodes Other invertebrates Fungi Plants Protists Eubacteria Archea Algae
Tissues;cells:	Fat Heart Skeletal muscle Nervous system Liver Kidney Lung;gill Islet cell;pancreas;thymus Endothelial;epithelial;mesothelial cell Blood cells Genital Other cell lines CHO HEK HeLa Neuroblastoma Fibroblast HUVEC Macrophage-derived Lymphocyte Platelet Stem cells
Preparations:	SMP Intact organism Intact organ Permeabilized cells Permeabilized tissue Homogenate Isolated mitochondria Chloroplasts Enzyme Oxidase;biochemical oxidation Intact cells
Enzymes:	Complex I Adenine nucleotide translocase Complex II;succinate dehydrogenase Complex III Complex IV;cytochrome c oxidase Complex V;ATP synthase Inner mt-membrane transporter Marker enzyme Supercomplex TCA cycle and matrix dehydrogenases Uncoupling protein
Regulation:	pH Aerobic glycolysis ADP ATP ATP production AMP Calcium Coupling efficiency;uncoupling Cyt c Flux control Inhibitor Ion;substrate transport mt-Membrane potential Oxygen kinetics PCr;Cr Phosphate Redox state Substrate Temperature Threshold;excess capacity Uncoupler Fatty acid Amino acid Q-junction effect
Coupling:	ET LEAK ROUTINE OXPHOS
Pathways:	F N S Gp DQ CIV NS Other combinations ROX
High-resolution respirometry:	pH Oxygraph-2k TIP2k O2k-Fluorometer NO TPP Ca O2k-Spectrophotometer O2k-Manual O2k-Protocol Theory NextGen-O2k
Event:	A1 A2 A3 A4 B1 B2 B3 B4 C1 C2 C3 C4 D1 D2 D3 D4 E1 E2 Oral Poster P-flash Review
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Free text: Any additional information (e.g. affiliations of authors). == Affiliations == :::: 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 ==Reference== ::::* 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.

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