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Updated: Jun 28, 2026
Immunohistochemistry Test for the Lyssavirus Antigen Detection from Formalin-Fixed Tissues
Published on: October 26, 2021
Detection of viral RNA from paraffin-embedded tissues after prolonged formalin fixation
Michelle D McKinney1, Steven J Moon, David A Kulesh
1GEO-CENTERS, Inc., United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21702-5011, United States.
Background:
Isolating amplifiable RNA from formalin-fixed, paraffin-embedded (FFPE) tissues is more difficult than isolating DNA because of RNases, chemical modification of the RNA, and cross-linking of nucleic acids and proteins. Tissues containing infectious disease agents that require biosafety level (BSL)-3 and -4 necessitate fixation times of 21 and 30 days, respectively.
Objective:
To improve procedures for extracting RNA from these FFPE tissues and detect the RNA with the more sensitive TaqManbased reverse transcriptase (RT)-PCR.
Study Design:
Through a single modification of a commercially available kit, we were able to extract amplifiable RNA and detect West Nile virus (WNV), Marburg virus (MARV), and Ebola virus (EBOV)-infected tissues using TaqMan assays.
Results:
Formalin fixation results in an approximately 2log(10) reduction in detection limit when compared to fresh tissues. Increasing proteinase K digestion (24h) improved extraction of amplifiable RNA from FFPE tissues. The TaqMan results were comparable to more traditional detection results such as virus isolation.
Conclusion:
This improved extraction procedure for obtaining RNA combined with the TaqMan RT-PCR assays permit retrospective and prospective studies on FFPE tissues infected with BSL-3 and -4 pathogens.
Insights
Improved RNA extraction from formalin-fixed, paraffin-embedded (FFPE) tissues using a modified kit and TaqMan reverse transcriptase (RT)-PCR allows sensitive detection of high-risk viruses. This method is crucial for studying Biosafety Level 3 and 4 pathogens in FFPE samples.
Area of Science:
- Molecular biology
- Virology
- Pathogen detection
Background:
- RNA extraction from FFPE tissues is challenging due to RNA degradation and cross-linking.
- Longer fixation times for Biosafety Level (BSL)-3 and -4 pathogens exacerbate RNA isolation difficulties.
Purpose of the Study:
- To enhance RNA extraction from FFPE tissues.
- To improve detection sensitivity using TaqMan-based reverse transcriptase (RT)-PCR.
Main Methods:
- A single modification was applied to a commercial RNA extraction kit.
- TaqMan assays were utilized for sensitive detection of viral RNA.
Main Results:
- The modified procedure successfully extracted amplifiable RNA from FFPE tissues.
- Formalin fixation reduced RNA detection limits by approximately 2log(10) compared to fresh tissues.
- Extended proteinase K digestion (24 hours) improved RNA yield and quality.
Conclusions:
- The optimized RNA extraction and TaqMan RT-PCR assay enable reliable detection of BSL-3 and -4 pathogens in FFPE tissues.
- This method facilitates retrospective and prospective studies on FFPE samples infected with high-containment viruses.
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