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Updated: Jun 13, 2026
Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction
Published on: August 21, 2016
Improving molecular detection of fungal DNA in formalin-fixed paraffin-embedded tissues: comparison of five tissue
C Muñoz-Cadavid1, S Rudd, S R Zaki
1Mycotic Diseases Branch, Centers for Disease Control and Prevention, Atlanta, Georgia, USA.
Abstract:
DNA extraction from formalin-fixed paraffin-embedded (FFPE) tissues is difficult and requires special protocols in order to extract small amounts of DNA suitable for amplification. Most described methods report an amplification success rate between 60 and 80%; therefore, there is a need to improve molecular detection and identification of fungi in FFPE tissue. Eighty-one archived FFPE tissues with a positive Gomori methenamine silver (GMS) stain were evaluated using five different commercial DNA extraction kits with some modifications. Three different panfungal PCR assays were used to detect fungal DNA, and two housekeeping genes were used to assess the presence of amplifiable DNA and to detect PCR inhibitors. The sensitivities of the five extraction protocols were compared, and the quality of DNA detection (calculated for each kit as the number of housekeeping gene PCR-positive samples divided by the total number of samples) was 60 to 91% among the five protocols. The efficiencies of the three different panfungals used (calculated as the number of panfungal-PCR-positive samples divided by the number of housekeeping gene PCR-positive samples) were 58 to 93%. The panfungal PCR using internal transcribed spacer 3 (ITS3) and ITS4 primers yielded a product in most FFPE tissues. Two of the five DNA extraction kits (from TaKaRa and Qiagen) showed similar and promising results. However, one method (TaKaRa) could extract fungal DNA from 69 of the 74 FFPE tissues from which a housekeeping gene could be amplified and was also cost-effective, with a nonlaborious protocol. Factors such as sensitivity, cost, and labor will help guide the selection of the most appropriate method for the needs of each laboratory.
Insights
Optimizing DNA extraction from formalin-fixed paraffin-embedded (FFPE) tissues is crucial for fungal detection. The TaKaRa kit demonstrated superior performance in extracting fungal DNA from FFPE samples, proving cost-effective and efficient.
Area of Science:
- Molecular Biology
- Mycology
- Histopathology
Background:
- DNA extraction from formalin-fixed paraffin-embedded (FFPE) tissues presents challenges due to DNA fragmentation.
- Existing methods for fungal DNA extraction from FFPE tissues have variable success rates (60-80%), necessitating improvements for accurate molecular detection.
- Gomori methenamine silver (GMS) staining is a common method for visualizing fungi in FFPE tissues, but molecular confirmation is often desired.
Purpose of the Study:
- To evaluate and compare the efficiency of five commercial DNA extraction kits for fungal DNA recovery from FFPE tissues.
- To assess the quality of extracted DNA using PCR amplification of housekeeping genes and identify potential PCR inhibitors.
- To determine the optimal DNA extraction method for sensitive and reliable molecular identification of fungi in FFPE samples.
Main Methods:
- Eighty-one GMS-positive FFPE tissue samples were processed using five different commercial DNA extraction kits with modifications.
- Three distinct panfungal polymerase chain reaction (PCR) assays were employed for fungal DNA detection.
- Two housekeeping genes were amplified to evaluate DNA quality and detect PCR inhibitors.
Main Results:
- DNA extraction quality, assessed by housekeeping gene PCR, ranged from 60% to 91% across the five kits.
- Panfungal PCR efficiencies varied from 58% to 93%, with the ITS3/ITS4 primer set showing broad utility.
- The TaKaRa DNA extraction kit demonstrated high efficiency (69/74 samples) and cost-effectiveness, with a non-laborious protocol, alongside promising results from Qiagen kits.
Conclusions:
- The TaKaRa DNA extraction kit is a highly effective, cost-efficient, and user-friendly option for isolating fungal DNA from FFPE tissues.
- Selection of an appropriate DNA extraction method should consider sensitivity, cost, and laboratory workflow.
- Improved DNA extraction protocols are vital for enhancing the molecular detection and identification of fungi in FFPE samples.
