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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 expressionGene expression (GEX), 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 4PlexMultiplexing4Plex, 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.

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

  • 实验方案

  • 发表日期：

    2026年

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  • Heidi Pharo,
  • Sara Brandt-Winge,
  • Evy Marie Thorkildsen,
  • Hege Marie Vedeld,
  • Guro E. Lind

摘要

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 expressionGene expression (GEX), 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 4PlexMultiplexing4Plex, 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.