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Real-Time PCR Quantification of Heteroplasmy in a Mouse Model with Mitochondrial DNA of C57BL/6 and NZB/BINJ Strains

Mouse models are widely employed to study mitochondrial inheritance, which have implications to several human diseases caused by mutations in the mitochondrial genome (mtDNA). These mouse models take advantage of polymorphisms between the mtDNA of the NZB/BINJ and the mtDNA of common inbred laborato...

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Autores principales: Machado, Thiago Simões, Macabelli, Carolina Habermann, Sangalli, Juliano Rodrigues, Rodrigues, Thiago Bittencourt, Smith, Lawrence Charles, Meirelles, Flávio Vieira, Chiaratti, Marcos Roberto
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4537288/
https://www.ncbi.nlm.nih.gov/pubmed/26274500
http://dx.doi.org/10.1371/journal.pone.0133650
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author Machado, Thiago Simões
Macabelli, Carolina Habermann
Sangalli, Juliano Rodrigues
Rodrigues, Thiago Bittencourt
Smith, Lawrence Charles
Meirelles, Flávio Vieira
Chiaratti, Marcos Roberto
author_facet Machado, Thiago Simões
Macabelli, Carolina Habermann
Sangalli, Juliano Rodrigues
Rodrigues, Thiago Bittencourt
Smith, Lawrence Charles
Meirelles, Flávio Vieira
Chiaratti, Marcos Roberto
author_sort Machado, Thiago Simões
collection PubMed
description Mouse models are widely employed to study mitochondrial inheritance, which have implications to several human diseases caused by mutations in the mitochondrial genome (mtDNA). These mouse models take advantage of polymorphisms between the mtDNA of the NZB/BINJ and the mtDNA of common inbred laboratory (i.e., C57BL/6) strains to generate mice with two mtDNA haplotypes (heteroplasmy). Based on PCR followed by restriction fragment length polymorphism (PCR-RFLP), these studies determine the level of heteroplasmy across generations and in different cell types aiming to understand the mechanisms underlying mitochondrial inheritance. However, PCR-RFLP is a time-consuming method of low sensitivity and accuracy that dependents on the use of restriction enzyme digestions. A more robust method to measure heteroplasmy has been provided by the use of real-time quantitative PCR (qPCR) based on allelic refractory mutation detection system (ARMS-qPCR). Herein, we report an ARMS-qPCR assay for quantification of heteroplasmy using heteroplasmic mice with mtDNA of NZB/BINJ and C57BL/6 origin. Heteroplasmy and mtDNA copy number were estimated in germline and somatic tissues, providing evidence of the reliability of the approach. Furthermore, it enabled single-step quantification of heteroplasmy, with sensitivity to detect as low as 0.1% of either NZB/BINJ or C57BL/6 mtDNA. These findings are relevant as the ARMS-qPCR assay reported here is fully compatible with similar heteroplasmic mouse models used to study mitochondrial inheritance in mammals.
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spelling pubmed-45372882015-08-20 Real-Time PCR Quantification of Heteroplasmy in a Mouse Model with Mitochondrial DNA of C57BL/6 and NZB/BINJ Strains Machado, Thiago Simões Macabelli, Carolina Habermann Sangalli, Juliano Rodrigues Rodrigues, Thiago Bittencourt Smith, Lawrence Charles Meirelles, Flávio Vieira Chiaratti, Marcos Roberto PLoS One Research Article Mouse models are widely employed to study mitochondrial inheritance, which have implications to several human diseases caused by mutations in the mitochondrial genome (mtDNA). These mouse models take advantage of polymorphisms between the mtDNA of the NZB/BINJ and the mtDNA of common inbred laboratory (i.e., C57BL/6) strains to generate mice with two mtDNA haplotypes (heteroplasmy). Based on PCR followed by restriction fragment length polymorphism (PCR-RFLP), these studies determine the level of heteroplasmy across generations and in different cell types aiming to understand the mechanisms underlying mitochondrial inheritance. However, PCR-RFLP is a time-consuming method of low sensitivity and accuracy that dependents on the use of restriction enzyme digestions. A more robust method to measure heteroplasmy has been provided by the use of real-time quantitative PCR (qPCR) based on allelic refractory mutation detection system (ARMS-qPCR). Herein, we report an ARMS-qPCR assay for quantification of heteroplasmy using heteroplasmic mice with mtDNA of NZB/BINJ and C57BL/6 origin. Heteroplasmy and mtDNA copy number were estimated in germline and somatic tissues, providing evidence of the reliability of the approach. Furthermore, it enabled single-step quantification of heteroplasmy, with sensitivity to detect as low as 0.1% of either NZB/BINJ or C57BL/6 mtDNA. These findings are relevant as the ARMS-qPCR assay reported here is fully compatible with similar heteroplasmic mouse models used to study mitochondrial inheritance in mammals. Public Library of Science 2015-08-14 /pmc/articles/PMC4537288/ /pubmed/26274500 http://dx.doi.org/10.1371/journal.pone.0133650 Text en © 2015 Machado et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Machado, Thiago Simões
Macabelli, Carolina Habermann
Sangalli, Juliano Rodrigues
Rodrigues, Thiago Bittencourt
Smith, Lawrence Charles
Meirelles, Flávio Vieira
Chiaratti, Marcos Roberto
Real-Time PCR Quantification of Heteroplasmy in a Mouse Model with Mitochondrial DNA of C57BL/6 and NZB/BINJ Strains
title Real-Time PCR Quantification of Heteroplasmy in a Mouse Model with Mitochondrial DNA of C57BL/6 and NZB/BINJ Strains
title_full Real-Time PCR Quantification of Heteroplasmy in a Mouse Model with Mitochondrial DNA of C57BL/6 and NZB/BINJ Strains
title_fullStr Real-Time PCR Quantification of Heteroplasmy in a Mouse Model with Mitochondrial DNA of C57BL/6 and NZB/BINJ Strains
title_full_unstemmed Real-Time PCR Quantification of Heteroplasmy in a Mouse Model with Mitochondrial DNA of C57BL/6 and NZB/BINJ Strains
title_short Real-Time PCR Quantification of Heteroplasmy in a Mouse Model with Mitochondrial DNA of C57BL/6 and NZB/BINJ Strains
title_sort real-time pcr quantification of heteroplasmy in a mouse model with mitochondrial dna of c57bl/6 and nzb/binj strains
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4537288/
https://www.ncbi.nlm.nih.gov/pubmed/26274500
http://dx.doi.org/10.1371/journal.pone.0133650
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