Comparison of methods for the quantification of cell-free DNA isolated from cell culture supernatant.
Comparison of methods for the quantification of cell-free DNA isolated from cell culture supernatant.
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DOI：
10.1177/1010428319866369
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发表时间：
2019-08-01
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影响因子：
--
通讯作者：
Holdenrieder, Stefan
中科院分区：
文献类型：
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作者：
Bronkhorst, Abel Jacobus;Ungerer, Vida;Holdenrieder, Stefan
更好地了解无细胞DNA的生物学特性是开发具有临床意义的无细胞DNA检测方法的重要一步。由于血液样本的巨大异质性使体内无细胞DNA的表征变得复杂，因此正在进行越来越多的体外细胞培养实验，这些实验提供了更高水平的控制。然而，细胞培养研究目前面临着三个值得注意的警告。首先，体外无细胞DNA的浓度相对较低。其次，不同细胞类型的培养基中游离DNA的中位数和大小差异很大。第三，单个细胞系培养基中游离DNA的数量和大小随时间而波动。虽然这些都是有趣的发现，但它也可能是实验混乱的一个重要来源，并强调了方法优化和标准化的重要性。因此，在本研究中，我们比较了五种常用的无细胞DNA定量方法，包括定量聚合酶链反应、Qubit双链DNA高灵敏度测定法、Quant-iT PicoGreen测定法、Bioanalyzer高灵敏度DNA测定法和NanoDrop Onec。对结果数据的分析，以及对理论值的解释（即各自方法的理论检测和定量极限），可以计算出与每种定量方法和不同细胞类型相关的几个重要分析前步骤的最佳条件，包括(1)应收集培养基进行无细胞DNA提取的时间点；(2)用于分离无细胞DNA的细胞培养上清量，(3)洗脱缓冲液的体积，(4)用于定量的无细胞DNA样品的体积。
Gaining a better understanding of the biological properties of cell-free DNA constitutes an important step in the development of clinically meaningful cell-free DNA-based tests. Since the in vivo characterization of cell-free DNA is complicated by the immense heterogeneity of blood samples, an increasing number of in vitro cell culture experiments, which offer a greater level of control, are being conducted. However, cell culture studies are currently faced with three notable caveats. First, the concentration of cell-free DNA in vitro is relatively low. Second, the median amount and size of cell-free DNA in culture medium varies greatly between cell types. Third, the amount and size of cell-free DNA in the culture medium of a single cell line fluctuates over time. Although these are interesting findings, it can also be a great source of experimental confusion and emphasizes the importance of method optimization and standardization. Therefore, in this study, we compared five commonly used cell-free DNA quantification methods, including quantitative polymerase chain reaction, Qubit Double-Stranded DNA High Sensitivity assay, Quant-iT PicoGreen Assay, Bioanalyzer High Sensitivity DNA assay, and NanoDrop Onec. Analysis of the resulting data, along with an interpretation of theoretical values (i.e. the theoretical detection and quantification limits of the respective methods), enables the calculation of optimal conditions for several important preanalytical steps pertaining to each quantification method and different cell types, including the (1) time-point at which culture medium should be collected for cell-free DNA extraction, (2) amount of cell culture supernatant from which to isolate cell-free DNA, (3) volume of elution buffer, and (4) volume of cell-free DNA sample to use for quantification.