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Cells
Volume 10
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10.3390/cells10040819
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Article

Quantification of Circulating Cell Free Mitochondrial DNA in Extracellular Vesicles with PicoGreen™ in Liquid Biopsies: Fast Assessment of Disease/Trauma Severity

by
Michela Marcatti
1,
Jamal Saada
2,
Ikenna Okereke
3,
Charles E. Wade
4,
Stefan H. Bossmann
5,
Massoud Motamedi
6 and
Bartosz Szczesny
2,6,*
1
Department of Neurology, University of Texas Medical Branch, Galveston, TX 77555, USA
2
Department of Anesthesiology, University of Texas Medical Branch, Galveston, TX 77555, USA
3
Department of Surgery, University of Texas Medical Branch, Galveston, TX 77555, USA
4
Department of Surgery, University of Texas Health Science Center in Houston, Houston, TX 77003, USA
5
Department of Cancer Biology, The University of Kansas Medical Center, Kansas City, KS 66160, USA
6
Departments of Ophthalmology and Visual Sciences, University of Texas Medical Branch, Galveston, TX 77555, USA
*
Author to whom correspondence should be addressed.
Cells 2021, 10(4), 819; https://doi.org/10.3390/cells10040819
Submission received: 4 March 2021 / Revised: 1 April 2021 / Accepted: 2 April 2021 / Published: 6 April 2021
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Abstract

The analysis of circulating cell free DNA (ccf-DNA) is an emerging diagnostic tool for the detection and monitoring of tissue injury, disease progression, and potential treatment effects. Currently, most of ccf-DNA in tissue and liquid biopsies is analysed with real-time quantitative PCR (qPCR) that is primer- and template-specific, labour intensive and cost-inefficient. In this report we directly compare the amounts of ccf-DNA in serum of healthy volunteers, and subjects presenting with various stages of lung adenocarcinoma, and survivors of traumatic brain injury using qPCR and quantitative PicoGreen™ fluorescence assay. A significant increase of ccf-DNA in lung adenocarcinoma and traumatic brain injury patients, in comparison to the group of healthy human subjects, was found using both analytical methods. However, the direct correlation between PicoGreen™ fluorescence and qPCR was found only when mitochondrial DNA (mtDNA)-specific primers were used. Further analysis of the location of ccf-DNA indicated that the majority of DNA is located within lumen of extracellular vesicles (EVs) and is easily detected with mtDNA-specific primers. We have concluded that due to the presence of active DNases in the blood, the analysis of DNA within EVs has the potential of providing rapid diagnostic outcomes. Moreover, we speculate that accurate and rapid quantification of ccf-DNA with PicoGreen™ fluorescent probe used as a point of care approach could facilitate immediate assessment and treatment of critically ill patients.
Keywords: circulating cell free DNA; traumatic brain injury; trauma severity; extracellular veciscles; PicoGreenTM staining; mitochondrial DNA circulating cell free DNA; traumatic brain injury; trauma severity; extracellular veciscles; PicoGreenTM staining; mitochondrial DNA

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MDPI and ACS Style

Marcatti, M.; Saada, J.; Okereke, I.; Wade, C.E.; Bossmann, S.H.; Motamedi, M.; Szczesny, B. Quantification of Circulating Cell Free Mitochondrial DNA in Extracellular Vesicles with PicoGreen™ in Liquid Biopsies: Fast Assessment of Disease/Trauma Severity. Cells 2021, 10, 819. https://doi.org/10.3390/cells10040819

AMA Style

Marcatti M, Saada J, Okereke I, Wade CE, Bossmann SH, Motamedi M, Szczesny B. Quantification of Circulating Cell Free Mitochondrial DNA in Extracellular Vesicles with PicoGreen™ in Liquid Biopsies: Fast Assessment of Disease/Trauma Severity. Cells. 2021; 10(4):819. https://doi.org/10.3390/cells10040819

Chicago/Turabian Style

Marcatti, Michela, Jamal Saada, Ikenna Okereke, Charles E. Wade, Stefan H. Bossmann, Massoud Motamedi, and Bartosz Szczesny. 2021. "Quantification of Circulating Cell Free Mitochondrial DNA in Extracellular Vesicles with PicoGreen™ in Liquid Biopsies: Fast Assessment of Disease/Trauma Severity" Cells 10, no. 4: 819. https://doi.org/10.3390/cells10040819

APA Style

Marcatti, M., Saada, J., Okereke, I., Wade, C. E., Bossmann, S. H., Motamedi, M., & Szczesny, B. (2021). Quantification of Circulating Cell Free Mitochondrial DNA in Extracellular Vesicles with PicoGreen™ in Liquid Biopsies: Fast Assessment of Disease/Trauma Severity. Cells, 10(4), 819. https://doi.org/10.3390/cells10040819

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