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(Reference retrieved automatically from Web of Science through information on FAPESP grant and its corresponding number as mentioned in the publication by the authors.)

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

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Machado, Thiago Simoes [1, 2, 3] ; Macabelli, Carolina Habermann [1, 3] ; Sangalli, Juliano Rodrigues [2, 3] ; Rodrigues, Thiago Bittencourt [1] ; Smith, Lawrence Charles [2, 4] ; Meirelles, Flavio Vieira [2, 3] ; Chiaratti, Marcos Roberto [1, 2, 3]
Total Authors: 7
[1] Univ Fed Sao Carlos, Ctr Ciencias Biol & Saude, Dept Genet & Evolucao, BR-13565905 Sao Carlos, SP - Brazil
[2] Univ Sao Paulo, Fac Med Vet & Zootecnia, Dept Cirurgia, BR-05508270 Sao Paulo, SP - Brazil
[3] Univ Sao Paulo, Fac Zootecnia & Engn Alimentos, Dept Med Vet, BR-13635900 Pirassununga, SP - Brazil
[4] Univ Montreal, Fac Med Vet, Ctr Rech Reprod Anim, St Hyacinthe, PQ J2S 7C6 - Canada
Total Affiliations: 4
Document type: Journal article
Source: PLoS One; v. 10, n. 8 AUG 14 2015.
Web of Science Citations: 5

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. (AU)

FAPESP's process: 12/50231-6 - Molecular basis of mitochondrial inheritance: the role of mitochondrial fusion
Grantee:Marcos Roberto Chiaratti
Support Opportunities: Research Grants - Young Investigators Grants
FAPESP's process: 10/13384-3 - Effect of introducing embryonic or somatic mitochondria on development and on mitochondrial inheritance in cattle
Grantee:Flávio Vieira Meirelles
Support Opportunities: Regular Research Grants
FAPESP's process: 10/09561-7 - Effect of introducing a large amount of embryonic or somatic mitochondria on development and on mitochondrial inheritance in bovines
Grantee:Marcos Roberto Chiaratti
Support Opportunities: Scholarships in Brazil - Post-Doctorate
FAPESP's process: 12/12951-7 - Transplantation of cytoplasm subjected to oxidative stress as a model for study of mitochondrial disease inheritance
Grantee:Thiago Simões Machado
Support Opportunities: Scholarships in Brazil - Master