TY - JOUR
T1 - Quantification of circulating cell free mitochondrial dna in extracellular vesicles with picogreen™ in liquid biopsies
T2 - Fast assessment of disease/trauma severity
AU - Marcatti, Michela
AU - Saada, Jamal
AU - Okereke, Ikenna
AU - Wade, Charles E.
AU - Bossmann, Stefan H.
AU - Motamedi, Massoud
AU - Szczesny, Bartosz
N1 - Publisher Copyright:
© 2021 by the authors. Licensee MDPI, Basel, Switzerland.
PY - 2021/4
Y1 - 2021/4
N2 - 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. Cur-rently, 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 adeno-carcinoma and traumatic brain injury patients, in comparison to the group of healthy human sub-jects, 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.
AB - 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. Cur-rently, 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 adeno-carcinoma and traumatic brain injury patients, in comparison to the group of healthy human sub-jects, 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.
KW - Circulating cell free DNA
KW - Extracellular vecis-cles
KW - PicoGreen™ staining
KW - Trauma severity
KW - Traumatic brain injury
UR - http://www.scopus.com/inward/record.url?scp=85105219373&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85105219373&partnerID=8YFLogxK
U2 - 10.3390/cells10040819
DO - 10.3390/cells10040819
M3 - Article
C2 - 33917426
AN - SCOPUS:85105219373
SN - 2073-4409
VL - 10
JO - Cells
JF - Cells
IS - 4
M1 - 819
ER -