Abstract
BackgroundDroplet 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.MethodsTwo 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.ResultsA 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.ConclusionImplementation 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.
Highlights
Droplet digital Polymerase chain reaction (PCR) allows absolute quantification of nucleic acids and has potential for improved non-invasive detection of Deoxyribonucleic acid (DNA) methylation
We have developed a robust internal control for methylationspecific Droplet digital PCR (ddPCR), the 4Plex
In conclusion, the 4Plex internal control increases the precision of methylation-specific ddPCR analyses by reducing the variability in methylation concentrations, correcting for variable input amount, and by reducing the effect of chromosomal aberrations
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. Digital PCR (dPCR) enables absolute quantification of nucleic acids. Technology development during the last decade has led to several commercial systems for dPCR, resulting in a rapid increase in the publication rate of dPCR studies [2, 3]. With the dPCR technology, a PCR mixture can be randomly divided into a large number of partitions. One of the most commonly used platforms is the droplet digital PCR (ddPCR), where the partitions are represented by thousands of nanoliter-scale droplets, formed by water-in-oil emulsion [4,5,6]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
