Towards standardisation of cell-free DNA measurement in plasma: controls for extraction efficiency, fragment size bias and quantification.
Towards standardisation of cell-free DNA measurement in plasma: controls for extraction efficiency, fragment size bias and quantification.
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DOI：
10.1007/s00216-014-7835-3
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发表时间：
2014-10
影响因子：
4.3
通讯作者：
Huggett, Jim F.
中科院分区：
文献类型：
--
作者：
Devonshire, Alison S.;Whale, Alexandra S.;Gutteridge, Alice;Jones, Gerwyn;Cowen, Simon;Foy, Carole A.;Huggett, Jim F.
关键词：
循环中的游离DNA(CfDNA)正成为产前检测、癌症诊断和癌症监测的重要临床分析物质。提取阶段对于确保测量少数核酸组分的分析方法的临床灵敏度至关重要，例如以母体为主的cfDNA中来自胎儿的序列。因此，为了验证cfDNA方法的有效性，需要进行质量控制来测量提取效率、片段大小偏差和产量。我们通过一项研究，将三种特定的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血迷你试剂盒进行比较，评估了外部DNA尖峰用于监测这些参数的有效性。我们发现，各试剂盒的提取效率顺序为CNA Kit>；DBM Kit>；NS Kit>；FA Kit，且CNA和NS试剂盒比DBM试剂盒能更好地显示提取液中较小的DNA片段。我们通过比较血浆样本中七种不同参考基因检测的定量PCR测量结果，并用数字聚合酶链式反应验证这些方法，研究了改进cfDNA产量报告的方法。我们注意到，基于对一些目标基因(例如TERT)的测量得出的cfDNA数量平均比其他检测方法(例如erv3)高出两倍以上。我们的结论是，使用GeNorm方法对多个参考基因进行分析和平均，可以更可靠地估计cfDNA总量。用于血浆细胞游离DNA定量的单个和多个参考基因标准化的比较本文的在线版本(DOI：10.1007/s002160147835-3)包含补充材料，可供授权用户使用。
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. Comparison of single and multiple reference gene normalisation for quantification of plasma cell free DNA The online version of this article (doi:10.1007/s00216-014-7835-3) contains supplementary material, which is available to authorized users.
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影响因子：
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