循环细胞外DNA（cfDNA）中的DNA甲基化生物标志物在癌症管理中具有巨大的临床潜力。大多数DNA甲基化分析方法需要亚硫酸盐转化，导致DNA降解和丢失。对于自然断裂且通常数量较低的cfDNA来说，这尤其具有挑战性。本研究旨在确定一个最佳的cfDNA分离和亚硫酸盐转化试剂盒的组合，以用于后续分析血浆中DNA甲基化生物标志物的实验。在经过测试的五个亚硫酸盐转化试剂盒（EpiJET亚硫酸盐转化试剂盒，EpiTect Plus DNA亚硫酸盐试剂盒（EpiTect），EZ DNA甲基化直接试剂盒，Imprint DNA修饰试剂盒（Imprint）和Premium亚硫酸盐试剂盒）中，EpiTect试剂盒和Imprint试剂盒分别产生了最高和最低的DNA产量和回收率，EpiTect试剂盒的DNA量超过Imprint试剂盒的两倍。在经过测试的三个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约是另外两个试剂盒的两倍，但也伴有更多的高分子量DNA。比较各种cfDNA分离试剂盒和亚硫酸盐转化试剂盒的组合时，CNA试剂盒和EpiTect试剂盒被确定为表现最佳的组合，通过液滴数字PCR（ddPCR）测量，其从正常血浆中产生了最高的亚硫酸盐转化cfDNA收率。作为原则性证明，该试剂盒组合用于处理13个结直肠癌患者的血浆样本，以进行后续的BCAT1和IKZF1的ddPCR甲基化分析。在10例（60%）第四期患者和3例（33%）第三期患者中，检测到BCAT1和/或IKZF1的甲基化。根据对五个亚硫酸盐转化试剂盒和三个cfDNA分离试剂盒以及它们的组合进行全面评估，CNA试剂盒和EpiTect试剂盒被确定为最佳组合，具有在一系列DNA输入量下最高的DNA产量和回收率。该组合已成功用于从癌症患者的血浆中检测临床相关的DNA甲基化生物标志物。©2023年BioMed Central Ltd., Springer Nature旗下的一部分。

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.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.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.© 2023. BioMed Central Ltd., part of Springer Nature.