EDTA-mediated inhibition of DNases protects circulating cell-free DNA from ex vivo degradation in blood samples
EDTA-mediated inhibition of DNases protects circulating cell-free DNA from ex vivo degradation in blood samples
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DOI：
10.1016/j.clinbiochem.2015.02.014
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发表时间：
2015-10-01
影响因子：
2.8
通讯作者：
Soares Costa, Sandra Santana
中科院分区：
文献类型：
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作者：
Barra, Gustavo Barcelos;Santa Rita, Ticiane Henriques;Soares Costa, Sandra Santana
目的：血浆-EDTA和血清中的胞外DNA是一个越来越受关注的生物标志物，特别是在产前诊断和肿瘤学中。本研究的目的是以无细胞胎儿DNA(Ccff)NA为工具，比较这些标本中的DNase活性，并探讨其对循环中无细胞DNA产量(CcfDNA)的影响。设计和方法：采用基于qPCR降解探针的内源性DNase活性测定方法，研究外源核酸酶的作用，以及不同温度条件下ccffDNA的降解情况。在所有情况下，所有男性ccffDNA都通过针对Y染色体特异序列DYS-14的qPCR进行定量。此外，在内源性DNA酶活性测定之前，在非抗凝的血浆和血清中加入一系列稀释的EDTA，以研究EDTA对血液DNA的抑制作用。结果：血清中的内源核酸酶活性是EDTA血浆的14.9倍。DNase I处理不改变EDTA血浆中ccffDNA的产量，但使其在血清中完全降解。在未抗凝的血浆和血清中加入剂量增加的EDTA，可使其核酸酶活性逐渐受到抑制。结论：外源性和内源性DNA酶在血清中的活性更高，抗凝剂EDTA间接抑制血液中的DNA酶，从而保护ccfDNA免受EDTA血浆中血液DNA酶分析前的影响。0 2015年，作者。加拿大临床化学家协会。由爱思唯尔公司出版。这是一篇基于CC by License(http://creativecommons.org/licenses/by/4.0/).的开放获取文章
Objetives: The extracellular DNA occurring in plasma-EDTA and serum is a biomarker of growing interest, especially in prenatal diagnosis and oncology. The objectives of the present study were to compare the DNase activity in these specimens and to investigate its ex-vivo impact over the circulating cell-free DNA yield (ccfDNA), using the circulating cell-free fetal DNA (ccff)NA) as a tool.Design and methods: EDTA-plasma and serum from women bearing male fetus were submitted to an endogenous DNase activity assay based on qPCR hydrolysis probe degradation, they were treated with DNAse I to investigate the action of an exogenous nuclease and also submitted to different temperature conditions to investigate the temperature-dependent degradation of the ccffDNA. In all instances, all male ccffDNA were quantified by qPCR targeting the Y chromosome-specific sequence DYS-14. Moreover, a serial dilution of EDTA was added to nonanticoagulated plasma and serum before the endogenous DNAse activity assay, to investigate the EDTA-mediated inhibition of the blood's DNase.Results: The endogenous nuclease activity was 14.9-fold higher in serum compared to EDTA-plasma. The DNAse I treatment did not alter the ccffDNA yields in EDTA-plasma, but completely degraded it in serum. The addition of increasing doses of EDTA to nonanticoagulated plasma and serum resulted in a stepwise inhibition of their nucleases activity. Finally, we observed a much more pronounced temperature-mediated decrease on the ccffDNA amount in serum compared to EDTA-plasma.Conclusion: The exogenous and endogenous DNases are more active in serum, the anticoagulant EDTA indirectly inhibits blood DNases, and consequently ccfDNA is protected from the blood's DNase preanalytical impact in EDTA-plasma. 0 2015 The Authors. The Canadian Society of Clinical Chemists. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).