Department of Molecular Medicine, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran.
Department of Gynecology & Obstetric, Shohada Tajrish Educational Hospital, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
J Assist Reprod Genet. 2019 May;36(5):1029-1038. doi: 10.1007/s10815-019-01425-w. Epub 2019 Feb 28.
Methylated cell-free fetal DNA (cffDNA) in maternal plasma can potentially be used as a biomarker for accurate noninvasive prenatal testing (NIPT) of fetal disorders. Recovery and purification of cffDNA are key steps for downstream applications. In this study, we aimed to developed and evaluated different aspects of an optimized method and compared its efficiency with common methods used for extraction of methylated cffDNA.
Single factor experiments, Plackett-Burman (PB) design, and response surface methodology (RSM) were conducted for conventional Triton/Heat/Phenol (cTHP) method optimization. The total cell-free DNA (cfDNA) was extracted from pooled maternal plasma using the optimized method called the Triton/Heat/Phenol/Glycogen (THPG), cTHP method, a column-based kit, and a magnetic bead-based kit. In the next step, methylated cfDNA from the extracted total cfDNA was enriched using a methylated DNA immunoprecipitation (MeDIP) kit. Real-time quantitative polymerase chain reaction was performed on the RASSF1 gene and hyper region to determine the genomic equivalents per milliliter (GEq/ml) values of the methylated cfDNA and cffDNA, respectively.
The optimum values of the significant factors affecting cfDNA extraction from 200 μl of plasma were 3% SDS, 1% Triton X-100, 0.9 μg/μl glycogen, and 0.3 M sodium acetate. The GEq/ml values of methylated cffDNA extracted using the THPG method were significantly higher than for the tested extraction methods (p < 0.001).
Our results indicate that the THPG method is more efficient than the other tested methods for extraction of low copy number methylated cffDNA from a small volume of maternal plasma.
孕妇血浆中的游离甲基化胎儿 DNA（cffDNA）可作为胎儿疾病无创产前检测（NIPT）的生物标志物。cffDNA 的回收和纯化是下游应用的关键步骤。本研究旨在开发和评估优化方法的不同方面，并将其与常用的提取甲基化 cffDNA 的方法进行比较。
采用单因素实验、Plackett-Burman（PB）设计和响应面法（RSM）对传统的 Triton/Heat/Phenol（cTHP）方法进行优化。采用优化后的 Triton/Heat/Phenol/Glycogen（THPG）法、cTHP 法、柱式试剂盒和磁珠试剂盒从混合孕妇血浆中提取总游离 DNA（cfDNA）。下一步，使用甲基化 DNA 免疫沉淀（MeDIP）试剂盒从提取的总 cfDNA 中富集甲基化 cfDNA。采用实时定量聚合酶链反应（PCR）检测 RASSF1 基因和超区，以确定甲基化 cfDNA 和 cffDNA 的基因组当量数（GEq/ml）值。
影响从 200 μl 血浆中提取 cfDNA 的显著因素的最佳值为 3% SDS、1% Triton X-100、0.9 μg/μl 糖原和 0.3 M 醋酸钠。THPG 法提取的甲基化 cffDNA 的 GEq/ml 值明显高于其他测试方法（p<0.001）。
本研究结果表明，与其他测试方法相比，THPG 法更有效，可从小体积孕妇血浆中提取低拷贝数甲基化 cffDNA。
