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Methylation Specific Multiplex Droplet PCR using Polymer Droplet Generator Device for Hematological Diagnostics
Published on: June 29, 2020
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A robust internal control for high-precision DNA methylation analyses by droplet digital PCR
Heidi D Pharo1,2,3,4, Kim Andresen1,2,3, Kaja C G Berg1,2,3
11Department of Molecular Oncology, Institute for Cancer Research, Oslo University Hospital, the Norwegian Radium Hospital, PO Box 4950, Nydalen, NO-0424 Oslo, Norway.
Clinical Epigenetics
|February 28, 2018
Summary
A new 4Plex internal control and PoDCall algorithm significantly improve DNA methylation analysis precision using droplet digital PCR (ddPCR). This enhances non-invasive detection accuracy for clinical applications.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- Droplet digital PCR (ddPCR) offers absolute nucleic acid quantification with potential for enhanced non-invasive DNA methylation detection.
- Developing a robust internal control is crucial for improving the precision of methylation-specific ddPCR analyses.
Purpose of the Study:
- To develop and validate a robust internal control for precise methylation analysis using ddPCR.
- To enhance the accuracy and reliability of non-invasive DNA methylation detection.
Main Methods:
- Two control design strategies were evaluated: gene family targeting and multi-locus pericentromeric targeting.
- Assay performance was optimized and evaluated on the Bio-Rad QX200 ddPCR platform using 34 colorectal cancer cell lines.
- A 4Plex panel (EPHA3, KBTBD4, PLEKHF1, SYT10) was identified as the optimal control and tested with methylation assays for CDO1, SEPT9, and VIM.
Main Results:
- The 4Plex panel demonstrated superior performance as an internal control for ddPCR methylation analysis.
- Normalization with the 4Plex control reduced variability, corrected for template input differences, and mitigated the impact of chromosomal aberrations.
- A novel R-based algorithm, Positive Droplet Calling (PoDCall), was developed for standardized and consistent ddPCR threshold determination.
Conclusions:
- The implementation of the 4Plex internal control and PoDCall algorithm significantly enhances the precision of DNA methylation analysis via ddPCR.
- These advancements improve the reliability of non-invasive DNA methylation detection, with implications for diagnostics and research.
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