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

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...

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Bibliographic Details
Published in:Clinical epigenetics 2018-02, Vol.10 (1), p.24-24, Article 24
Main Authors: Pharo, Heidi D, Andresen, Kim, Berg, Kaja C G, Lothe, Ragnhild A, Jeanmougin, Marine, Lind, Guro E
Format: Article
Language:English
Subjects:
4Plex
Algorithms
Caco-2 Cells
Cell Line, Tumor
Colorectal Neoplasms - genetics
Cysteine Dioxygenase - genetics
Digital PCR
DNA Methylation
HCT116 Cells
HT29 Cells
Humans
Internal control
Methodology
Methylation
Normalization
Nucleic acids
PoDCall
Polymerase chain reaction
Real-Time Polymerase Chain Reaction - standards
Reference Standards
Septins - genetics
Vimentin - genetics
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Summary: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.
ISSN:1868-7075
1868-7083
DOI:10.1186/s13148-018-0456-5