Stine H Kresse, Sara Brandt-Winge, Heidi Pharo, Bjørnar T B Flatin, Marine Jeanmougin, Hege Marie Vedeld, Guro E Lind
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The aim of the present study was to identify an optimal combination of cfDNA isolation and bisulfite conversion kits for downstream analysis of DNA methylation biomarkers in plasma.</p><p><strong>Results: </strong>Of the five tested bisulfite conversion kits (EpiJET Bisulfite Conversion Kit, EpiTect Plus DNA Bisulfite Kit (EpiTect), EZ DNA Methylation-Direct Kit, Imprint DNA Modification Kit (Imprint) and Premium Bisulfite Kit), the highest and lowest DNA yield and recovery were achieved using the EpiTect kit and the Imprint kit, respectively, with more than double the amount of DNA for the EpiTect kit. Of the three tested cfDNA isolation kits (Maxwell RSC ccfDNA Plasma Kit, QIAamp Circulating Nucleic Acid Kit (CNA) and QIAamp MinElute ccfDNA Mini Kit), the CNA kit yielded around twice as much cfDNA compared to the two others kits, although with more high molecular weight DNA present. When comparing various combinations of cfDNA isolation kits and bisulfite conversion kits, the CNA kit and the EpiTect kit were identified as the best-performing combination, resulting in the highest yield of bisulfite converted cfDNA from normal plasma, as measured by droplet digital PCR (ddPCR). As a proof of principle, this kit combination was used to process plasma samples from 13 colorectal cancer patients for subsequent ddPCR methylation analysis of BCAT1 and IKZF1. Methylation of BCAT1 and/or IKZF1 was identified in 6/10 (60%) stage IV patients and 1/3 (33%) stage III patients.</p><p><strong>Conclusions: </strong>Based on a thorough evaluation of five bisulfite conversion kits and three cfDNA isolation kits, both individually and in combination, the CNA kit and the EpiTect kit were identified as the best-performing kit combination, with highest DNA yield and recovery across a range of DNA input amounts. 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引用次数: 0
摘要
背景:循环无细胞DNA(cfDNA)中的DNA甲基化生物标志物在癌症治疗中具有巨大的临床潜力。大多数DNA甲基化分析方法都需要亚硫酸氢盐转化,从而导致DNA降解和丢失。这对cfDNA来说尤其具有挑战性,因为cfDNA是天然碎片化的,通常以少量存在。本研究的目的是确定cfDNA分离和亚硫酸氢盐转化试剂盒的最佳组合,用于血浆中DNA甲基化生物标志物的下游分析。结果:在五种测试的亚硫酸氢盐转化试剂盒(EpiJET亚硫酸氢转化试剂盒、EpiTect Plus DNA亚硫酸氢试剂盒(EpiTect)、EZ DNA甲基化直接试剂盒、印迹DNA修饰试剂盒(印迹)和高级亚硫酸氢化试剂盒)中,使用EpiTect试剂盒和印迹试剂盒分别获得了最高和最低的DNA产率和回收率,其DNA量是EpiTect试剂盒的两倍以上。在三种测试的cfDNA分离试剂盒(Maxwell RSC ccfDNA血浆试剂盒、QIAamp循环核酸试剂盒(CNA)和QIAamp MinElute ccfDNA迷你试剂盒)中,CNA试剂盒产生的cfDNA大约是其他两种试剂盒的两倍,尽管存在更多的高分子量DNA。当比较cfDNA分离试剂盒和亚硫酸氢盐转化试剂盒的各种组合时,CNA试剂盒和EpiTect试剂盒被确定为性能最佳的组合,通过液滴数字PCR(ddPCR)测量,从正常血浆中获得亚硫酸氢氢盐转化的cfDNA的产率最高。作为原理证明,该试剂盒组合用于处理13名癌症结直肠癌患者的血浆样本,用于随后对BCAT1和IKZF1进行ddPCR甲基化分析。在6/10(60%)的IV期患者和1/3(33%)的III期患者中发现BCAT1和/或IKZF1的甲基化。结论:根据对五个亚硫酸氢盐转化试剂盒和三个cfDNA分离试剂盒(单独和组合)的全面评估,CNA试剂盒和EpiTect试剂盒被确定为性能最佳的试剂盒组合,在一系列DNA输入量中具有最高的DNA产量和回收率。该组合成功用于检测癌症患者血浆中临床相关的DNA甲基化生物标志物。
Evaluation of commercial kits for isolation and bisulfite conversion of circulating cell-free tumor DNA from blood.
Background: DNA methylation biomarkers in circulating cell-free DNA (cfDNA) have great clinical potential for cancer management. Most methods for DNA methylation analysis require bisulfite conversion, causing DNA degradation and loss. This is particularly challenging for cfDNA, which is naturally fragmented and normally present in low amounts. The aim of the present study was to identify an optimal combination of cfDNA isolation and bisulfite conversion kits for downstream analysis of DNA methylation biomarkers in plasma.
Results: Of the five tested bisulfite conversion kits (EpiJET Bisulfite Conversion Kit, EpiTect Plus DNA Bisulfite Kit (EpiTect), EZ DNA Methylation-Direct Kit, Imprint DNA Modification Kit (Imprint) and Premium Bisulfite Kit), the highest and lowest DNA yield and recovery were achieved using the EpiTect kit and the Imprint kit, respectively, with more than double the amount of DNA for the EpiTect kit. Of the three tested cfDNA isolation kits (Maxwell RSC ccfDNA Plasma Kit, QIAamp Circulating Nucleic Acid Kit (CNA) and QIAamp MinElute ccfDNA Mini Kit), the CNA kit yielded around twice as much cfDNA compared to the two others kits, although with more high molecular weight DNA present. When comparing various combinations of cfDNA isolation kits and bisulfite conversion kits, the CNA kit and the EpiTect kit were identified as the best-performing combination, resulting in the highest yield of bisulfite converted cfDNA from normal plasma, as measured by droplet digital PCR (ddPCR). As a proof of principle, this kit combination was used to process plasma samples from 13 colorectal cancer patients for subsequent ddPCR methylation analysis of BCAT1 and IKZF1. Methylation of BCAT1 and/or IKZF1 was identified in 6/10 (60%) stage IV patients and 1/3 (33%) stage III patients.
Conclusions: Based on a thorough evaluation of five bisulfite conversion kits and three cfDNA isolation kits, both individually and in combination, the CNA kit and the EpiTect kit were identified as the best-performing kit combination, with highest DNA yield and recovery across a range of DNA input amounts. The combination was successfully used for detection of clinically relevant DNA methylation biomarkers in plasma from cancer patients.
Clinical EpigeneticsBiochemistry, Genetics and Molecular Biology-Developmental Biology
CiteScore
8.90
自引率
5.30%
发文量
150
审稿时长
12 weeks
期刊介绍:
Clinical Epigenetics, the official journal of the Clinical Epigenetics Society, is an open access, peer-reviewed journal that encompasses all aspects of epigenetic principles and mechanisms in relation to human disease, diagnosis and therapy. Clinical trials and research in disease model organisms are particularly welcome.