1Department of Molecular Oncology, Institute for Cancer Research, Oslo University Hospital, the Norwegian Radium Hospital, PO Box 4950, Nydalen, NO-0424 Oslo, Norway.
2KG Jebsen Colorectal Cancer Research Centre, Oslo University Hospital, Oslo, Norway.
Clin Epigenetics. 2018 Feb 21;10:24. doi: 10.1186/s13148-018-0456-5. eCollection 2018.
Droplet digital PCR (ddPCR) allows absolute quantification of nucleic acids and has potential for improved non-invasive detection of DNA methylation. For increased precision of the methylation analysis, we aimed to develop a robust internal control for use in methylation-specific ddPCR.
Two control design approaches were tested: (a) targeting a genomic region shared across members of a gene family and (b) combining multiple assays targeting different pericentromeric loci on different chromosomes. Through analyses of 34 colorectal cancer cell lines, the performance of the control assay candidates was optimized and evaluated, both individually and in various combinations, using the QX200™ droplet digital PCR platform (Bio-Rad). The best-performing control was tested in combination with assays targeting methylated , , and .
A 4Plex panel consisting of , , , and was identified as the best-performing control. The use of the 4Plex for normalization reduced the variability in methylation values, corrected for differences in template amount, and diminished the effect of chromosomal aberrations. Positive Droplet Calling (PoDCall), an R-based algorithm for standardized threshold determination, was developed, ensuring consistency of the ddPCR results.
Implementation of a robust internal control, i.e., the 4Plex, and an algorithm for automated threshold determination, PoDCall, in methylation-specific ddPCR increase the precision of DNA methylation analysis.
液滴数字 PCR（ddPCR）允许对核酸进行绝对定量，并且具有改进的非侵入性检测 DNA 甲基化的潜力。为了提高甲基化分析的精度，我们旨在开发一种稳健的内参，用于甲基化特异性 ddPCR。
测试了两种控制设计方法：（a）针对基因家族成员共有的基因组区域，（b）结合针对不同染色体上不同着丝粒区域的多个检测。通过对 34 个结直肠癌细胞系的分析，使用 QX200™液滴数字 PCR 平台（Bio-Rad）优化并评估了候选控制检测的性能，单独使用和组合使用。对表现最佳的控制进行了测试，与针对甲基化、、和的检测相结合。
确定由、、和组成的 4plex 面板为表现最佳的控制。使用 4plex 进行归一化可降低甲基化值的变异性，校正模板数量的差异，并减少染色体异常的影响。开发了一种基于 R 的标准化阈值确定算法 Positive Droplet Calling（PoDCall），以确保 ddPCR 结果的一致性。
在甲基化特异性 ddPCR 中实施稳健的内部对照（即 4plex）和用于自动阈值确定的算法 PoDCall 可提高 DNA 甲基化分析的精度。
