Plasma-derived cell-free mitochondrial DNA: A novel non-invasive methodology to identify mitochondrial DNA haplogroups in humans

Newell, Christopher; Hume, Stacey; Greenway, Steven C.; Podemski, Lynn; Shearer, Jane; Khan, Aneal

doi:10.1016/j.ymgme.2018.10.002

Cited by 23 publications

(19 citation statements)

References 31 publications

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“…However, it was assumed that the circular nature of mitochondrial DNA delays its degradation by circulating nucleases, so the presence of intact mitochondria was not suspected and the structural characteristics associated with McfDNA were not investigated. 35 In addition to observe by fractionation that a great part of mitochondrial DNA is associated to high size structures, we initially found striking differences between McfDNA and NcfDNA in respect to stability and size distribution.…”

Section: Discussionmentioning

confidence: 76%

“…Intact full mitochondrial genomes were also observed in the cell free DNA fraction of both healthy individuals and patients with mitochondrial disease. However, it was assumed that the circular nature of mitochondrial DNA delays its degradation by circulating nucleases, so the presence of intact mitochondria was not suspected and the structural characteristics associated with McfDNA were not investigated …”

Section: Discussionmentioning

confidence: 99%

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Blood contains circulating cell‐free respiratory competent mitochondria

et al. 2020

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Mitochondria are considered as the power‐generating units of the cell due to their key role in energy metabolism and cell signaling. However, mitochondrial components could be found in the extracellular space, as fragments or encapsulated in vesicles. In addition, this intact organelle has been recently reported to be released by platelets exclusively in specific conditions. Here, we demonstrate for the first time, that blood preparation with resting platelets, contains whole functional mitochondria in normal physiological state. Likewise, we show, that normal and tumor cultured cells are able to secrete their mitochondria. Using serial centrifugation or filtration followed by polymerase chain reaction‐based methods, and Whole Genome Sequencing, we detect extracellular full‐length mitochondrial DNA in particles over 0.22 µm holding specific mitochondrial membrane proteins. We identify these particles as intact cell‐free mitochondria using fluorescence‐activated cell sorting analysis, fluorescence microscopy, and transmission electron microscopy. Oxygen consumption analysis revealed that these mitochondria are respiratory competent. In view of previously described mitochondrial potential in intercellular transfer, this discovery could greatly widen the scope of cell‐cell communication biology. Further steps should be developed to investigate the potential role of mitochondria as a signaling organelle outside the cell and to determine whether these circulating units could be relevant for early detection and prognosis of various diseases.

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Section: Discussionmentioning

confidence: 76%

Section: Discussionmentioning

confidence: 99%

Blood contains circulating cell‐free respiratory competent mitochondria

et al. 2020

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“…Among the variables that we had identified from other studies, the volume in which DNA was eluted, which ranged from 30 to 200 µL, was not consistent and could potentially be a significant determinant of yields (8,22,25,31,(33)(34)(35)(36)(37). To determine if all the DNA was being eluted from the MB using our current volume (60 µL), we eluted DNA in both 60 and 120 µL of ES.…”

Section: Optimization Of Extraction Parametersmentioning

confidence: 90%

“…Based on the variability between protocols used in published studies, we identified protease digestion, detergent solubilization, sample volume, and incubation temperature and duration as potential factors that might impact ccf-DNA recovery (8,22,25,31,(33)(34)(35)(36)(37). Therefore, we tested these variables for both ccf-mtDNA and ccf-nDNA (total ccf-DNA) recovery by testing concentrations, volume, and time to confirm and refine the effects on ccf-DNA yields (Fig.…”

Section: Optimization Of Digestion Parametersmentioning

confidence: 99%

An automated, high throughput methodology optimized for quantitative cell-free mitochondrial and nuclear DNA isolation from plasma

Ware

Desai

Lopez

et al. 2020

Preprint

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Circulating, cell-free mitochondrial DNA (ccf-mtDNA) and nuclear DNA (ccf-nDNA) are under investigation as biomarkers for various diseases. Optimal ccf-mtDNA isolation parameters, like those outlined for ccf-nDNA, have not been established. Here, we optimized a protocol for both ccf-mtDNA and ccf-nDNA recovery using a magnetic bead-based isolation process on an automated 96-well platform. Using the optimized protocol, our data show 6-fold improved yields of ccf-mtDNA when compared to the starting protocol. Digestion conditions, liquid handling characteristics, and magnetic particle processor programming all contributed to increased recovery and improved reproducibility. To our knowledge, this is the first high-throughput approach optimized for mtDNA and nDNA recovery and serves as an important starting point for clinical studies.Graphical Abstract

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“…Based on the variability between protocols used in published studies, we identified protease digestion, detergent solubilization, sample volume, and incubation temperature and duration as potential factors that might impact ccf-DNA recovery (8,22,25,30,(38)(39)(40)(41)(42). Therefore, we tested these variables for both ccf-mtDNA and ccf-nDNA (total ccf-DNA) recovery by testing concentrations, volume, and time to confirm and refine the effects on ccf-DNA yields (Fig.…”

Section: Optimization Of Digestion Parametersmentioning

confidence: 99%

An automated, high-throughput methodology optimized for quantitative cell-free mitochondrial and nuclear DNA isolation from plasma

Ware

Desai

Lopez

et al. 2020

Journal of Biological Chemistry

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Progress in the study of circulating, cell-free nuclear DNA (ccf-nDNA) in cancer detection has led to the development of non-invasive clinical diagnostic tests and has accelerated the evaluation of ccf-nDNA abundance as a disease biomarker. Likewise, circulating, cell-free mitochondrial DNA (ccf-mtDNA) is under similar investigation. However, optimal ccf-mtDNA isolation parameters have not been established, and inconsistent protocols for ccf-nDNA collection, storage, and analysis have hindered its clinical utility. Until now, no studies have established a method for high throughput isolation that considers both ccf-nDNA and ccf-mtDNA. We initially optimized human plasma digestion and extraction conditions for maximal recovery of these DNAs using a magnetic bead-based isolation method. However, when we incorporated this method onto a high throughput platform, initial experiments found that DNA isolated from identical human plasma samples displayed plate edge effects resulting in low ccf-mtDNA reproducibility, while ccf-nDNA was less affected. Therefore, we developed a detailed protocol optimized for both ccf-mtDNA and ccf-nDNA recovery that uses a magnetic bead-based isolation process on an automated 96-well platform. Overall, we calculate an improved efficiency of recovery of ~95-fold for ccf-mtDNA and 20-fold for ccf-nDNA when compared to the initial procedure. Digestion conditions, liquid handling characteristics, and magnetic particle processor programming all contributed to increased recovery and without detectable positional effects. To our knowledge, this is the first high throughput approach optimized for ccf-mtDNA and ccf-nDNA recovery and serves as an important starting point for clinical studies.

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Plasma-derived cell-free mitochondrial DNA: A novel non-invasive methodology to identify mitochondrial DNA haplogroups in humans

Cited by 23 publications

References 31 publications

Blood contains circulating cell‐free respiratory competent mitochondria

Blood contains circulating cell‐free respiratory competent mitochondria

An automated, high throughput methodology optimized for quantitative cell-free mitochondrial and nuclear DNA isolation from plasma

An automated, high-throughput methodology optimized for quantitative cell-free mitochondrial and nuclear DNA isolation from plasma

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