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Clin Epigenetics. 2018 Feb 21;10:24. doi: 10.1186/s13148-018-0456-5. eCollection 2018.

A robust internal control for high-precision DNA methylation analyses by droplet digital PCR.

Pharo HD1,2,3,4, Andresen K1,2,3, Berg KCG1,2,3, Lothe RA1,2,3,4, Jeanmougin M1,2,3, Lind GE1,2,3,4.

Author information

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.
3Centre for Cancer Biomedicine, Faculty of Medicine, University of Oslo, Oslo, Norway.
4Department of Biosciences, The Faculty of Mathematics and Natural Sciences, University of Oslo, Oslo, Norway.



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 CDO1, SEPT9, and VIM.


A 4Plex panel consisting of EPHA3, KBTBD4, PLEKHF1, and SYT10 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.


4Plex; Digital PCR; Internal control; Methylation; Normalization; PoDCall

Conflict of interest statement

Not applicableNot applicableThe authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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