Rausch Sebastian, Hasinger Oliver, König Thomas, Schlegel Anne, Weiss Gunter
Epigenomics AG, Berlin, Germany.
BMC Res Notes. 2019 Aug 30;12(1):551. doi: 10.1186/s13104-019-4595-3.
DNA methylation analysis via real-time PCR or other analytical techniques requires purified bisulfite converted DNA. We report on an automated high throughput solution for DNA extraction, bisulfite-conversion, and purification of 96 samples with an input volume of up to 3.5 mL of plasma or urine, using reagents from the commercially available Epi BisKit.
Magnetic bead-based DNA extraction, bisulfite conversion at high temperature, and efficient DNA purification was conducted on a customized commercially available liquid-handling platform. A highly interlaced 4 × 24 sample protocol was implemented for DNA extraction, elution in a 96-well plate, efficient bisulfite-conversion and extensive purification. The resulting bisulfite-converted DNA was stored in a 96-well format, ready for PCR set-up or other down-stream applications. The automated method is a walk-away solution for processing 96 samples in 7 h 30 min. Performance of the method was validated by comparison with the standard manual method of the Epi BiSKit using technical and biological samples. Overall DNA yield was assessed with a standardized β-actin assay. The automated workflow demonstrated equivalent performance to the manual method for technical, plasma and urine samples. It may provide a new standard for effective high-throughput preparation of bisulfite-converted DNA from a variety of high volume liquid biopsy specimens.
通过实时聚合酶链反应（PCR）或其他分析技术进行DNA甲基化分析需要纯化的亚硫酸氢盐转化DNA。我们报告了一种自动化的高通量解决方案，该方案使用市售Epi BisKit试剂，可对96个样本进行DNA提取、亚硫酸氢盐转化和纯化，血浆或尿液输入体积高达3.5 mL。
在定制的市售液体处理平台上进行基于磁珠的DNA提取、高温亚硫酸氢盐转化和高效DNA纯化。实施了高度交错的4×24样本方案用于DNA提取、在96孔板中洗脱、高效亚硫酸氢盐转化和广泛纯化。所得的亚硫酸氢盐转化DNA以96孔格式储存，随时可用于PCR设置或其他下游应用。该自动化方法是一种可在7小时30分钟内处理96个样本的无需值守的解决方案。通过使用技术样本和生物样本与Epi BiSKit的标准手动方法进行比较，验证了该方法的性能。使用标准化的β-肌动蛋白测定评估总体DNA产量。自动化工作流程在技术样本、血浆和尿液样本方面表现出与手动方法相当的性能。它可能为从各种大量液体活检样本中有效高通量制备亚硫酸氢盐转化DNA提供新的标准。
