Janovičová Ľubica, Konečná Barbora, Vlková Barbora, Celec Peter
Institute of Molecular Biomedicine, Faculty of Medicine, Comenius University, Bratislava, Slovakia.
Institute of Pathophysiology, Faculty of Medicine, Comenius University, Bratislava, Slovakia.
Bio Protoc. 2020 Aug 20;10(16):e3726. doi: 10.21769/BioProtoc.3726.
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.
细胞外DNA作为一种诊断生物标志物受到研究，同时由于其促炎特性，它也是多种疾病病理生理学中的一个相关因素。使用标准的DNA分离程序和专门的商业试剂盒，可以从血浆、尿液、唾液或其他生物流体中提取细胞外DNA。分离的样品制备很重要，冻融可能会影响提取的细胞外DNA量。随后的离心操作可从样品中去除细胞和细胞碎片，以获得真正的细胞外DNA。尤其是来自动物实验的少量样品常常是个问题，并且会影响DNA产量。非常短的片段（100 bp）在分离过程中可能会丢失，并且难以通过PCR进行定量。荧光法可评估所有染色的DNA片段。选择细胞外DNA定量方法至关重要，至少两种方法的组合是理想的。程序的标准化或至少在研究论文中的报告对于结果比较至关重要。
