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首页> 外文期刊>Analytical and bioanalytical chemistry >Towards standardisation of cell-free DNA measurement in plasma: controls for extraction efficiency, fragment size bias and quantification
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Towards standardisation of cell-free DNA measurement in plasma: controls for extraction efficiency, fragment size bias and quantification

机译：实现血浆中无细胞DNA测量的标准化：提取效率，片段大小偏差和定量的控制

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Circulating cell-free DNA (cfDNA) is becoming an important clinical analyte for prenatal testing, cancer diagnosis and cancer monitoring. The extraction stage is critical in ensuring clinical sensitivity of analytical methods measuring minority nucleic acid fractions, such as foetal-derived sequences in predominantly maternal cfDNA. Consequently, quality controls are required for measurement of extraction efficiency, fragment size bias and yield for validation of cfDNA methods. We evaluated the utility of an external DNA spike for monitoring these parameters in a study comparing three specific cfDNA extraction methods [QIAamp? circulating nucleic acid (CNA) kit, NucleoSpin? Plasma XS (NS) kit and FitAmp?plasma/serum DNA isolation (FA) kit] with the commonly used QIAamp DNA blood mini (DBM) kit.We found that the extraction efficiencies of the kits ranked in the order CNA kit > DBM kit > NS kit > FA kit, and the CNA and NS kits gave a better representation of smaller DNA fragments in the extract than the DBM kit. We investigated means of improved reporting of cfDNA yield by comparing quantitative PCR measurements of seven different reference gene assays in plasma samples and validating these with digital PCR. We noted that the cfDNA quantities based on measurement of some target genes (e.g. TERT) were, on average, more than twofold higher than those of other assays (e.g. ERV3). We conclude that analysis and averaging of multiple reference genes using a GeNorm approach gives a more reliable estimate of total cfDNA quantity
机译：循环中的无细胞DNA（cfDNA）正在成为产前检测，癌症诊断和癌症监测的重要临床分析物。提取阶段对于确保分析方法的临床敏感性至关重要，该方法可测量少数核酸片段，例如主要来自母体cfDNA的胎儿衍生序列。因此，需要质量控制来测量提取效率，片段大小偏差和产率，以验证cfDNA方法。我们在一项研究中比较了三种特定的cfDNA提取方法[QIAamp ?？循环核酸（CNA）试剂盒NucleoSpin？血浆XS（NS）试剂盒和FitAmp？血浆/血清DNA分离（FA）试剂盒]与常用的QIAamp DNA血液迷你（DBM）试剂盒。我们发现，该试剂盒的提取效率按CNA试剂盒> DBM试剂盒的顺序排列> NS试剂盒> FA试剂盒，而CNA和NS试剂盒比DBM试剂盒更好地代表了提取物中较小的DNA片段。我们通过比较血浆样品中7种不同参考基因测定的定量PCR测量并用数字PCR进行验证，研究了改进的cfDNA产量报告方法。我们注意到，基于对某些靶基因（例如TERT）的测量得出的cfDNA数量平均比其他分析（例如ERV3）高出两倍以上。我们得出结论，使用GeNorm方法对多个参考基因进行分析和平均可以得出更可靠的总cfDNA数量估计值

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