Diefenbach Russell J, Lee Jenny H, Kefford Richard F, Rizos Helen
Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Macquarie University, Sydney, NSW 2109, Australia; The Poche Centre, Melanoma Institute Australia, NSW 2065, Australia.
Department of Clinical Medicine, Faculty of Medicine and Health Sciences, Macquarie University, Sydney, NSW 2109, Australia; The Poche Centre, Melanoma Institute Australia, NSW 2065, Australia; Department of Medical Oncology, Crown Princess Mary Cancer Centre, Westmead and Blacktown Hospitals, Sydney, NSW 2145, Australia.
Cancer Genet. 2018 Dec;228-229:21-27. doi: 10.1016/j.cancergen.2018.08.005. Epub 2018 Aug 29.
Analysis of liquid biopsies and the identification of non-invasive biomarkers for the diagnosis and prognosis of solid tumors has grown exponentially over the last few years. This has led to an increasing number of commercial kits optimised for the purification of circulating free (cf) DNA and RNA/miRNA from biofluids such as plasma, serum and urine. To optimise and standardise current practices we sought to evaluate the performance of spin column-based and magnetic bead-based commercial kits. The following commercial cfDNA purification kits were analysed in this study: QIAamp circulating nucleic acid kit (Qiagen, Germany); Plasma/serum cell-free circulating DNA Purification midi kit (Norgen Biotek, Canada); QIAamp minElute ccfDNA mini kit (Qiagen); Maxwell RSC ccfDNA plasma kit (Promega, USA); MagMAX cell-free DNA isolation kit (Applied Biosystems, USA); and NextPrep-Mag cfDNA isolation kit (Bioo Scientific, USA). Extracted DNA from the plasma of healthy individuals, either nonspiked or spiked with DNA fragments or cfDNA, was evaluated for recovery using either a BioRad Experion or ddPCR analysis. This study represents the first to use a comprehensive size distribution of spiked-in DNA fragments to evaluate commercial cfDNA kits. The commonly used spin column-based Qiagen QIAamp circulating nucleic acid kit was found to be the most consistent performing kit across the two evaluation assays employed. The Qiagen QIAamp minElute ccfDNA mini kit represented the best performing magnetic bead-based kit and provides an alternative based on lower cost/sample with a simpler workflow than spin column-based kits.
在过去几年中，液体活检分析以及用于实体瘤诊断和预后的非侵入性生物标志物的鉴定呈指数级增长。这导致了越来越多的商业试剂盒，这些试剂盒经过优化，可用于从血浆、血清和尿液等生物流体中纯化循环游离（cf）DNA和RNA/miRNA。为了优化和规范当前的做法，我们试图评估基于离心柱和磁珠的商业试剂盒的性能。本研究分析了以下商业cfDNA纯化试剂盒：QIAamp循环核酸试剂盒（德国Qiagen公司）；血浆/血清游离循环DNA纯化中量试剂盒（加拿大Norgen Biotek公司）；QIAamp minElute ccfDNA微量试剂盒（Qiagen公司）；Maxwell RSC ccfDNA血浆试剂盒（美国Promega公司）；MagMAX游离DNA分离试剂盒（美国应用生物系统公司）；以及NextPrep-Mag cfDNA分离试剂盒（美国Bioo Scientific公司）。使用BioRad Experion或ddPCR分析评估从健康个体血浆中提取的DNA的回收率，这些血浆要么未加样，要么加了DNA片段或cfDNA。本研究首次使用加样DNA片段的全面大小分布来评估商业cfDNA试剂盒。在采用的两种评估方法中，常用的基于离心柱的Qiagen QIAamp循环核酸试剂盒被发现是性能最稳定的试剂盒。Qiagen QIAamp minElute ccfDNA微量试剂盒是性能最佳的基于磁珠的试剂盒，并且基于较低的成本/样本以及比基于离心柱的试剂盒更简单的工作流程提供了一种替代方案。
