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Effect of Antigen Retrieval on Genomic DNA From Immunodissected Samples
Donald J Johann1, Ik Jae Shin1, Adam Roberge2
1Winthrop P. Rockefeller Cancer Institute, University of Arkansas for Medical Sciences, Little Rock, Arkansas.
Summary
Antigen retrieval (AR) and immunohistochemistry (IHC) can damage DNA, but gene mutations are still detectable. DNA from immunostained slides is viable for genomic analysis using digital droplet PCR and next-generation sequencing.
Area of Science:
- Molecular Biology
- Genomics
- Histology
Background:
- Immunohistochemical (IHC) staining visualizes proteins in tissues for research and clinical use.
- Antigen retrieval (AR) enhances IHC but can degrade DNA, impacting downstream genomic analysis.
- IHC is increasingly used for cell isolation via microdissection techniques.
Purpose of the Study:
- To investigate the effects of common AR methods on DNA quality and quantity in immunodissected samples.
- To assess the feasibility of genomic mutation analysis from DNA recovered after AR and IHC processing.
Main Methods:
- Samples (clinical and xenograft) underwent heat-induced epitope retrieval (HIER) or protease digestion.
- DNA quality and quantity were assessed using Qubit, fragment analysis, qPCR, and digital droplet PCR (ddPCR).
- Library preparation and targeted sequencing were performed on recovered DNA.
Main Results:
- HIER improved IHC staining but caused significant DNA fragmentation and reduced yield.
- Neither AR method prevented ddPCR amplification of small amplicons.
- Gene mutations were successfully identified from immunodissected clinical samples using ddPCR and NGS.
Conclusions:
- DNA recovered from immunostained slides after standard AR and IHC processing is suitable for genomic mutation analysis.
- Digital droplet PCR and next-generation sequencing (NGS) short-read methods can successfully identify gene mutations from these samples.
- This study validates a method for combined IHC and genomic analysis from a single tissue sample.
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