DNA Methylation Analyses of Tissue Samples and Cell-Free DNA by Droplet Digital PCR

Heidi Pharo; Sara Brandt-Winge; Evy Marie Thorkildsen; Hege Marie Vedeld; Guro E Lind

doi:10.1007/978-1-0716-4767-7_12

DNA Methylation Analyses of Tissue Samples and Cell-Free DNA by Droplet Digital PCR

Methods Mol Biol. 2026:2969:179-193. doi: 10.1007/978-1-0716-4767-7_12.

Authors

Heidi Pharo¹, Sara Brandt-Winge¹, Evy Marie Thorkildsen¹, Hege Marie Vedeld¹, Guro E Lind^{2

3}

Affiliations

¹ Department of Molecular Oncology, Institute for Cancer Research, Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway.
² Department of Molecular Oncology, Institute for Cancer Research, Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway. guroli@uio.no.
³ Department of Biosciences, Faculty of Mathematics and Natural Sciences, University of Oslo, Oslo, Norway. guroli@uio.no.

PMID: 41073866
DOI: 10.1007/978-1-0716-4767-7_12

Abstract

Droplet digital PCR (ddPCR) allows highly sensitive detection of minimal amounts of a target of interest. The most common applications of ddPCR have traditionally been detection of genetic variants, copy number variations and gene expression, but the method has great potential also for DNA methylation analyses. We here describe an optimized and standardized protocol for methylation-specific ddPCR analyses, including use of an internal control; the 4Plex, and an automated algorithm for scoring of positive droplets; PoDCall. We also describe a protocol for bisulfite treatment using either of two kits from QIAGEN, utilizing cell-free DNA (cfDNA) or tissue samples as staring material. Implementation of these protocols across laboratories could contribute to more robust and consistent ddPCR methylation results.

Keywords: 4Plex; Bisulfite treatment; DNA methylation; Droplet digital PCR; Optimization; PoDCall; Protocol; Standardization; cfDNA.

MeSH terms

Cell-Free Nucleic Acids* / analysis
Cell-Free Nucleic Acids* / genetics
DNA Methylation*
Humans
Polymerase Chain Reaction* / methods

Substances

Cell-Free Nucleic Acids