Absolute quantitation of a heteroplasmic mitochondrial DNA deletion using a multiplex three-primer real-time PCR assay

Bobby G. Poe; Marian Navratil; Edgar A. Arriaga

doi:10.1016/j.ab.2006.12.035

Absolute quantitation of a heteroplasmic mitochondrial DNA deletion using a multiplex three-primer real-time PCR assay

Bobby G. Poe, Marian Navratil, Edgar A. Arriaga

Chemistry (Twin Cities)

Research output: Contribution to journal › Article › peer-review

19 Scopus citations

Abstract

Quantitation of wild-type and deleted mitochondrial DNA (mtDNA) coexisting within the same cell (a.k.a., heteroplasmy) is important in mitochondrial disease and aging. We report the development of a multiplex three-primer PCR assay that is capable of absolute quantitation of wild-type and deleted mtDNA simultaneously. Molecular beacons were designed to hybridize with either type of mtDNA molecule, allowing real-time detection during PCR amplification. The assay is specific and can detect down to six copies of mtDNA, making it suitable for single-cell analyses. The relative standard deviation in the threshold cycle number is approximately 0.6%. Heteroplasmy was quantitated in individual cytoplasmic hybrid cells (cybrids), containing a large mtDNA deletion, and bulk cell samples. Individual cybrid cells contained 100-2600 copies of wild-type mtDNA and 950-4700 copies of deleted mtDNA, and the percentage of heteroplasmy ranged from 43 ± 16 to 95 ± 16%. The average amount of total mtDNA was 3800 ± 1600 copies/cybrid cell, and the average percentage of heteroplasmy correlated well with the bulk cell sample. The single-cell analysis also revealed that heteroplasmy in individual cells is highly heterogeneous. This assay will be useful for monitoring clonal expansions of mtDNA deletions and investigating the role of heteroplasmy in cell-to-cell heterogeneity in cellular models of mitochondrial disease and aging.

Original language	English (US)
Pages (from-to)	193-200
Number of pages	8
Journal	Analytical Biochemistry
Volume	362
Issue number	2
DOIs	https://doi.org/10.1016/j.ab.2006.12.035
State	Published - Mar 15 2007

Bibliographical note

Funding Information:
This work was supported by the National Institutes of Health (NIH, R01-AG20866). B.G.P. acknowledges support from an NIH–Chemistry/Biology Interface Training Grant (GM08700). E.A.A. is supported by an NIH Career Award (1K02-AG21453). The authors thank Carlos Moraes (University of Miami) for providing the ΔH2-1 cybrid cell line.

Keywords

Heteroplasmy
Mitochondrial DNA quantitation
Molecular beacons
Real-time PCR

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abstract = "Quantitation of wild-type and deleted mitochondrial DNA (mtDNA) coexisting within the same cell (a.k.a., heteroplasmy) is important in mitochondrial disease and aging. We report the development of a multiplex three-primer PCR assay that is capable of absolute quantitation of wild-type and deleted mtDNA simultaneously. Molecular beacons were designed to hybridize with either type of mtDNA molecule, allowing real-time detection during PCR amplification. The assay is specific and can detect down to six copies of mtDNA, making it suitable for single-cell analyses. The relative standard deviation in the threshold cycle number is approximately 0.6%. Heteroplasmy was quantitated in individual cytoplasmic hybrid cells (cybrids), containing a large mtDNA deletion, and bulk cell samples. Individual cybrid cells contained 100-2600 copies of wild-type mtDNA and 950-4700 copies of deleted mtDNA, and the percentage of heteroplasmy ranged from 43 ± 16 to 95 ± 16%. The average amount of total mtDNA was 3800 ± 1600 copies/cybrid cell, and the average percentage of heteroplasmy correlated well with the bulk cell sample. The single-cell analysis also revealed that heteroplasmy in individual cells is highly heterogeneous. This assay will be useful for monitoring clonal expansions of mtDNA deletions and investigating the role of heteroplasmy in cell-to-cell heterogeneity in cellular models of mitochondrial disease and aging.",

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Absolute quantitation of a heteroplasmic mitochondrial DNA deletion using a multiplex three-primer real-time PCR assay

Abstract

Bibliographical note

Keywords

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