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

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.

Mitochondrial Extracellular Vesicles in CNS Disorders: New Frontiers in Understanding the Neurological Disorders of the Brain

“…However, so far, this is the only study of these EVs and much further work is needed to develop an in-depth knowledge of their functions during ASD. Similar to the ASD findings, analysis of EVs extracted from serum of subjects with non-penetrating traumatic brain injury (TBI) showed a significant increase in mtDNA in TBI patients compared to the control group ( Marcatti et al, 2021 ), and this also needs to be followed up with extensive functional studies.…”

New Perspectives on the Importance of Cell-Free DNA Biology

“…They range in size from 40 to 100 nm and are constitutively generated from late endosomes [ 290 , 291 , 292 , 293 ]. Numerous studies have reported the presence of single- and double-stranded DNA as well as mtDNA [ 294 , 295 , 296 ], transposable elements [ 297 ] and viral DNA [ 298 , 299 ] in association with sEVs. The primary localization of exosomal DNA is not clear, as studies have shown DNA to be located in the lumen and on the outer surface [ 300 , 301 , 302 ].…”

“…Interestingly, a recent study reported that exercise triggered an increase of DNA levels only on the outer surface [ 301 ]. While there is ample evidence of exosome-associated DNA, it remains a controversial topic as highly cited studies have reported evidence both for the association of DNA with exosomes [ 295 , 303 , 304 ] and no association of DNA with exosomes [ 305 ]. Apart from biological diversity among different exosome types, which are yet to be fully explored, there may be other reasons for the discrepant findings reported by studies, such as differences in sample handling and exosome isolation protocols [ 306 ].…”

Cell-Free DNA in the Pathogenesis and Therapy of Non-Infectious Inflammations and Tumors

“…According to the latest data [ 17 ], the normal human plasma cfDNA concentration can be as high as 500 ng/μL. In cases of advanced cancers [ 18 , 19 , 20 ], autoimmune [ 21 , 22 , 23 , 24 , 25 ], inflammatory [ 26 ], traumatic [ 27 , 28 ], post-transplantation [ 29 ] or infectious diseases [ 30 , 31 ] usually a more increased amount is detected. In addition, cfDNA levels could also be increased, due to vigorous physical exertion (such as intense sports, e.g., half marathon, ultramarathon, TRX exercises) [ 32 , 33 ] and pregnancy [ 34 ].…”