Mitochondrial DNA as a non-invasive biomarker: Accurate quantification using real time quantitative PCR without co-amplification of pseudogenes and dilution bias
| Autor: | Ana Rodriguez-de-Ledesma, Phil Cunningham, Abas H. Laftah, Afshan N. Malik, Rojeen Shahni |
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| Rok vydání: | 2011 |
| Předmět: |
Genetic Markers
Genetics mtDNA control region Mitochondrial DNA Nuclear gene Genome Human Cells Pseudogene Biophysics Cell Biology Biology DNA Mitochondrial Polymerase Chain Reaction Biochemistry Genome Human mitochondrial genetics Cell Line Nuclear DNA Genome Mitochondrial Humans Molecular Biology Gene Pseudogenes |
| Zdroj: | Biochemical and Biophysical Research Communications. 412:1-7 |
| ISSN: | 0006-291X |
| DOI: | 10.1016/j.bbrc.2011.06.067 |
| Popis: | Circulating mitochondrial DNA (MtDNA) is a potential non-invasive biomarker of cellular mitochondrial dysfunction, the latter known to be central to a wide range of human diseases. Changes in MtDNA are usually determined by quantification of MtDNA relative to nuclear DNA (Mt/N) using real time quantitative PCR. We propose that the methodology for measuring Mt/N needs to be improved and we have identified that current methods have at least one of the following three problems: (1) As much of the mitochondrial genome is duplicated in the nuclear genome, many commonly used MtDNA primers co-amplify homologous pseudogenes found in the nuclear genome; (2) use of regions from genes such as β-actin and 18S rRNA which are repetitive and/or highly variable for qPCR of the nuclear genome leads to errors; and (3) the size difference of mitochondrial and nuclear genomes cause a "dilution bias" when template DNA is diluted. We describe a PCR-based method using unique regions in the human mitochondrial genome not duplicated in the nuclear genome; unique single copy region in the nuclear genome and template treatment to remove dilution bias, to accurately quantify MtDNA from human samples. |
| Databáze: | OpenAIRE |
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