Laboratory of Medical Genetics, Medical Research Institute, Cheil General Hospital and Women's Healthcare Center, Seoul, South Korea.
Department of Obstetrics and Gynecology, Cheil General Hospital and Women's Healthcare Center, Dankook University College of Medicine, 1-19, Mookjung-dong, Chung-gu, Seoul, 100-380, South Korea.
J Assist Reprod Genet. 2018 Apr;35(4):637-641. doi: 10.1007/s10815-018-1114-8. Epub 2018 Feb 8.
Recently, fetal placenta-specific epigenetic regions (FSERs) have been identified for quantification of cell-free fetal DNA (cff-DNA) for non-invasive prenatal testing (NIPT). The aim of this study was to evaluate the efficiencies of a column-based kit and magnetic bead-based kit for quantification of methylated FSERs from maternal plasma.
Maternal plasma was extracted from normal pregnant women within the gestational age of 10~13 weeks (n = 24). Total cell-free DNA (cf-DNA) was extracted using a column-based kit and magnetic bead-based kit from the plasma of the same pregnant woman, respectively. Methylated FSERs were enriched from the extracted total cf-DNA using a methyl-CpG-binding domain-based protein method. The four FSERs were simultaneously quantified by multiplex real-time polymerase chain reaction.
Methylated FSERs were detected in all samples extracted from both kits. However, the amplification of FSERs showed significant differences in the extraction efficiency of methylated FSERs between the two extraction methods. The Ct values of methylated FSERs extracted using the column-based kit were significantly lower than those obtained using the magnetic bead-based kit (P < 0.001 for all FSERs). The quantity of methylated FSERs was significantly higher for extracted DNA using the column-based kit than that extracted using the magnetic bead-based kit (P < 0.001 for all FSERs). Time and cost for the process of extraction were similar for the column kit and magnetic bead-based kit.
Our findings demonstrate that the column-based kit was more effective than the magnetic bead-based kit for isolation of methylated FSERs from maternal plasma as assessed by FSER detection.
最近，已经鉴定出胎儿胎盘特异性表观遗传区域（FSERs），用于定量分析游离胎儿 DNA（cff-DNA）进行非侵入性产前检测（NIPT）。本研究旨在评估基于柱和磁珠的试剂盒用于定量分析母体血浆中甲基化 FSER 的效率。
从 10~13 周妊娠的正常孕妇中提取母体血浆（n=24）。分别使用基于柱和磁珠的试剂盒从同一孕妇的血浆中提取总游离 DNA（cf-DNA）。使用基于甲基化 CpG 结合域蛋白的方法从提取的总 cf-DNA 中富集甲基化 FSERs。通过多重实时聚合酶链反应同时定量四种 FSERs。
从两种试剂盒提取的所有样本中均检测到甲基化 FSERs。然而，FSERs 的扩增显示出两种提取方法在提取甲基化 FSERs 的效率方面存在显著差异。使用基于柱的试剂盒提取的甲基化 FSER 的 Ct 值明显低于使用磁珠试剂盒提取的（所有 FSERs 的 P 值均<0.001）。使用基于柱的试剂盒提取的甲基化 FSER 的数量明显高于使用磁珠试剂盒提取的（所有 FSERs 的 P 值均<0.001）。两种试剂盒的提取过程的时间和成本相似。
我们的研究结果表明，基于柱的试剂盒在 FSER 检测方面比磁珠试剂盒更有效地从母体血浆中分离甲基化 FSERs。
