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Plasmid-normalized quantification of relative mitochondrial DNA copy number
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 establish...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6194030/ https://www.ncbi.nlm.nih.gov/pubmed/30337569 http://dx.doi.org/10.1038/s41598-018-33684-5 |
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author | Fazzini, Federica Schöpf, Bernd Blatzer, Michael Coassin, Stefan Hicks, Andrew A. Kronenberg, Florian Fendt, Liane |
author_facet | Fazzini, Federica Schöpf, Bernd Blatzer, Michael Coassin, Stefan Hicks, Andrew A. Kronenberg, Florian Fendt, Liane |
author_sort | Fazzini, Federica |
collection | PubMed |
description | 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-RNA(Leu) 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. |
format | Online Article Text |
id | pubmed-6194030 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-61940302018-10-24 Plasmid-normalized quantification of relative mitochondrial DNA copy number Fazzini, Federica Schöpf, Bernd Blatzer, Michael Coassin, Stefan Hicks, Andrew A. Kronenberg, Florian Fendt, Liane Sci Rep Article 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-RNA(Leu) 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. Nature Publishing Group UK 2018-10-18 /pmc/articles/PMC6194030/ /pubmed/30337569 http://dx.doi.org/10.1038/s41598-018-33684-5 Text en © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. |
spellingShingle | Article Fazzini, Federica Schöpf, Bernd Blatzer, Michael Coassin, Stefan Hicks, Andrew A. Kronenberg, Florian Fendt, Liane Plasmid-normalized quantification of relative mitochondrial DNA copy number |
title | Plasmid-normalized quantification of relative mitochondrial DNA copy number |
title_full | Plasmid-normalized quantification of relative mitochondrial DNA copy number |
title_fullStr | Plasmid-normalized quantification of relative mitochondrial DNA copy number |
title_full_unstemmed | Plasmid-normalized quantification of relative mitochondrial DNA copy number |
title_short | Plasmid-normalized quantification of relative mitochondrial DNA copy number |
title_sort | plasmid-normalized quantification of relative mitochondrial dna copy number |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6194030/ https://www.ncbi.nlm.nih.gov/pubmed/30337569 http://dx.doi.org/10.1038/s41598-018-33684-5 |
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