Isolation and Quantification of Extracellular DNA from Biofluids

Ľubica Janovičová; Barbora Konečná; Barbora Vlková; Peter Celec

doi:10.21769/BioProtoc.3726

Isolation and Quantification of Extracellular DNA from Biofluids

Bio Protoc. 2020 Aug 20;10(16):e3726. doi: 10.21769/BioProtoc.3726.

Authors

Ľubica Janovičová¹, Barbora Konečná¹, Barbora Vlková¹, Peter Celec^{1

2

3}

Affiliations

¹ Institute of Molecular Biomedicine, Faculty of Medicine, Comenius University, Bratislava, Slovakia.
² Institute of Pathophysiology, Faculty of Medicine, Comenius University, Bratislava, Slovakia.
³ Department of Molecular Biology, Faculty of Natural Sciences, Comenius University, Bratislava, Slovakia.

Abstract

Extracellular DNA is studied as a diagnostic biomarker, but also as a factor involved in the pathophysiology of several diseases due to its pro-inflammatory properties. Extracellular DNA can be extracted from plasma, urine, saliva or other biofluids using standard DNA isolation procedures and specialized commercial kits. Sample preparation for isolation is important, freezing and thawing may affect the amount of extracellular DNA extracted. Subsequent centrifugations remove cells and cell debris from the samples to obtain true extracellular DNA. Small volume of samples especially from animal experiments is often an issue and it affects the DNA yield. Very short fragments ( 100 bp) can be lost during isolation and are difficult to quantify using PCR. Fluorometric methods asses all stained DNA fragments. Selecting the method for quantification of extracellular DNA is crucial and combination of at least two methods is ideal. Standardization of procedures or at least their reporting in research papers is of utmost importance for comparison of results.

Keywords: Cell-free DNA; Cell-free nucleic acids; Circulating plasma DNA; DNA extraction; Sample preparation.