Quantitation of DNA Copy Number in Individual Mitochondrial Particles by Capillary Electrophoresis

Navrátil, Marián; Poe, Bobby G.; Arriaga, Edgar A.

doi:10.1021/ac0709192

Cited by 25 publications

(32 citation statements)

References 50 publications

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“…Although we have not determined numbers of mtDNAs within individual nucleoids, the distribution of our volumetric data is Fig. 5A is remarkably similar to typical distributions of mtDNAs per nucleoid described by others (11,42,54). This correlation is substantiated by the conversion of volumes to genomes.…”

Section: Discussionsupporting

confidence: 84%

Superresolution Fluorescence Imaging of Mitochondrial Nucleoids Reveals Their Spatial Range, Limits, and Membrane Interaction

Brown

Tkachuk

Shtengel

et al. 2011

Molecular and Cellular Biology

225

197

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A fundamental objective in molecular biology is to understand how DNA is organized in concert with various proteins, RNA, and biological membranes. Mitochondria maintain and express their own DNA (mtDNA), which is arranged within structures called nucleoids. Their functions, dimensions, composition, and precise locations relative to other mitochondrial structures are poorly defined. Superresolution fluorescence microscopy techniques that exceed the previous limits of imaging within the small and highly compartmentalized mitochondria have been recently developed. We have improved and employed both two-and three-dimensional applications of photoactivated localization microscopy (PALM and iPALM, respectively) to visualize the core dimensions and relative locations of mitochondrial nucleoids at an unprecedented resolution. PALM reveals that nucleoids differ greatly in size and shape. Three-dimensional volumetric analysis indicates that, on average, the mtDNA within ellipsoidal nucleoids is extraordinarily condensed. Two-color PALM shows that the freely diffusible mitochondrial matrix protein is largely excluded from the nucleoid. In contrast, nucleoids are closely associated with the inner membrane and often appear to be wrapped around cristae or crista-like inner membrane invaginations. Determinations revealing high packing density, separation from the matrix, and tight association with the inner membrane underscore the role of mechanisms that regulate access to mtDNA and that remain largely unknown.

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Section: Discussionsupporting

confidence: 84%

Superresolution Fluorescence Imaging of Mitochondrial Nucleoids Reveals Their Spatial Range, Limits, and Membrane Interaction

Brown

Tkachuk

Shtengel

et al. 2011

Molecular and Cellular Biology

225

197

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“…While we find no association between mtDNA content and embryonic stress, our data does not refute the proposed concept that embryos actively increase mitochondrial function and energy output as a compensatory response to overcome strained conditions. The number of mtDNA copies per mitochondrion in human cells can vary widely between 0 to 15 (21)(22)(23), meaning that the number of mtDNA copies per cell does not necessarily correlate to number of mitochondrial organelles. At least one report has demonstrated that mtDNA copy is a poor biomarker for mitochondrial content (24).…”

Section: Discussionmentioning

confidence: 99%

Accurate Quantitation of Mitochondrial DNA Reveals Uniform Levels in Human Blastocysts Irrespective of Ploidy, Age, or Implantation Potential

Victor

Brake

Tyndall

et al. 2017

Obstetrical &Amp; Gynecological Survey

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Main Outcome Measure(s): For each sample the mtDNA value was divided by the nuclear DNA (nDNA) value and the result was further subjected to mathematical analysis tailored to the genetic makeup of the source embryo.Result(s): On average the mathematical correction factor changed the conventionally determined mtDNA score of a given blastocyst via NGS by 1.43% +/-1.59% (N=1396), with maximal adjustments of 17.42%, and via qPCR by 1.33% +/-8.08% (N=150), with maximal adjustments of 50.00%. Levels of mtDNA in euploid and aneuploid embryos showed a statistically insignificant difference by NGS (euploids N=775, aneuploids N=621) and by qPCR (euploids N=100, aneuploids N=50). Blastocysts derived from younger or older patients had comparable mtDNA levels by NGS ( N=874 age group N=514) and by qPCR ( N age group N=58). Viable blastocysts did not contain significantly different mtDNA levels compared to unviable blastocysts when analyzed by NGS (implanted N=101, nonimplanted N=140) and by qPCR (implanted N=49, non-implanted N=51). Conclusion(s):We recommend implementation of the correction factor calculation to laboratories evaluating mtDNA levels in embryos by NGS or qPCR. When applied to our in-house data, the calculation reveals that overall levels of mtDNA are largely equal between blastocysts stratified by ploidy, age, or implantation potential.

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“…Lower pH values were determined in the acidic organelles of drug-resistant cell lines. Another report described the absolute quantitation of mitochondrial DNA (mtDNA) copy number in individual mitochondrial particles (152). Mitochondria isolated from cells expressing mito-DsRed2 (i.e., mitochondria label) were treated with a DNA intercalator (i.e., PicoGreen) and then analyzed by CE with a dual LIF detector.…”

Section: Bioparticlesmentioning

confidence: 99%

Capillary Electrophoresis in Bioanalysis

2008

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Science reports ∼4800 hits on capillary electrophoresis (CE) including 410 reviews. Because of the prominence of CE techniques in bioanalysis, we decided make them the focus of this review and selected 200 hundred papers representing advances in this field. The selected papers cover advances in CE theory, instrumentation, and methodologies that are specific to various analytes of biological origin or relevance. The group of analytes includes nucleic acids, proteins and peptides, carbohydrates, lipids, single cells, and bioparticles. In addition, we have included advances in the use of CE to define functional assays or to investigate biomolecular interactions. The use of microfabricated devices for CE analysis was not included because this is already covered in other review. Technique Developments Separation SchemesDetermining the velocity of the electroosmotic flow (EOF) and how it changes during an electrophoretic separation is still an important research topic. A simple method for EOF measurements using so-called thermal marks was reported (1). Here, a tungsten filament caused punctual heating at the capillary wall and caused a perturbation in the electrolyte concentration. A sequence of these "thermal marks" then migrated with the EOF until each mark reached and was detected by a conductivity detector. The feasibility of using thermal marks as internal EOF standards in different separation systems was thereby demonstrated.Isoelectric focusing separates amphoteric analytes such as proteins or peptides by the differences in their isoelectric points. Most of the reports on capillary isoelectric focusing (cIEF) describe an initial focusing phase after which the focused zones are mobilized and detected. A dynamic cIEF method for protein analysis was reported (2). This technique made it possible to control each protein's position and focused width by moving the pH gradient within the capillary through manipulation of the electric fields. An important advantage of this approach is the capability of collecting focused analytes from the central section, suggesting that there may be great potential for introducing selectively focused proteins to a second separation dimension such as LC or CE.Micellar electrokinetic capillary chromatography (MEKC) is typically incompatible with electrospray mass spectrometry (ESI-MS) because the nonvolatile surfactants in the micellar phase result in complicated adduct formation and loss of sensitivity during the electrospray process and because the presence of the organic solvent needed for electrospray may cause instability in the micellar phase. These drawbacks were overcome by using synthetic polymeric surfactants that can work as a pseudostationary phase and provide stable electrospray (3). The polymeric surfactant was made by polymerizing three amino acidderived (L-leucinol, L-isoleucinol, L-valinol) sulfated chiral surfactants. These polymeric

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Quantitation of DNA Copy Number in Individual Mitochondrial Particles by Capillary Electrophoresis

Cited by 25 publications

References 50 publications

Superresolution Fluorescence Imaging of Mitochondrial Nucleoids Reveals Their Spatial Range, Limits, and Membrane Interaction

Superresolution Fluorescence Imaging of Mitochondrial Nucleoids Reveals Their Spatial Range, Limits, and Membrane Interaction

Accurate Quantitation of Mitochondrial DNA Reveals Uniform Levels in Human Blastocysts Irrespective of Ploidy, Age, or Implantation Potential

Capillary Electrophoresis in Bioanalysis

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