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Updated: May 24, 2026
DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses
Published on: March 26, 2011
Enhanced fungal DNA-extraction from formalin-fixed, paraffin-embedded tissue specimens by application of thermal
Volker Rickerts1, Prasanna D Khot, Daisy L Ko
1Robert Koch Institut, Berlin, Germany. RickertsV@rki.de
Abstract:
Determining the etiology of invasive fungal infections (IFI) is critical for patient management as fungi vary in their susceptibility to antifungals. However, the etiology remains obscure in many cases due to negative culture results. The identification of fungal DNA by PCR in pathology blocks and sequencing it is an alternative approach to determine the cause of IFI. Previous studies identified fungal DNA in only 50% of samples with positive histopathology results, probably due to DNA damage by tissue fixation. We used realtime PCR to quantify human and fungal DNA from formalin-fixed, paraffin-embedded tissue specimens in order to study the effect of thermal energy during extraction on the yield of amplifiable DNA and subsequent identification of fungal DNA. Tissue sections from eight patients with proven IFI were subjected to DNA extraction with varying exposure to thermal energy. Amplifiable DNA increased up to 76-fold by increasing the incubation temperature from 65°C to 90°C and an additional increase was documented by incubating samples for up to 6 hours at this temperature. The augmented amplification of fungal DNA was associated with improved species identification by the sequencing of the PCR amplicons. This may help illuminate the etiology of IFI and thereby improve patient management by guiding antifungal therapy.
Insights
Optimizing DNA extraction using heat improves the detection of fungal DNA in tissue samples. This enhanced method aids in identifying the cause of invasive fungal infections (IFI) for better patient treatment.
Area of Science:
- Medical Mycology
- Molecular Diagnostics
- Pathology
Background:
- Accurate diagnosis of invasive fungal infections (IFI) is crucial for effective antifungal therapy.
- Culture methods often yield negative results, obscuring the etiology of IFI.
- Molecular methods like PCR offer an alternative but are limited by DNA degradation in fixed tissues.
Purpose of the Study:
- To investigate the impact of thermal energy during DNA extraction on the yield of amplifiable fungal DNA from formalin-fixed, paraffin-embedded (FFPE) tissues.
- To enhance the identification of fungal species in FFPE samples for improved IFI diagnostics.
Main Methods:
- Real-time PCR was used to quantify human and fungal DNA in FFPE tissue sections from eight patients with proven IFI.
- DNA extraction protocols were modified with varying incubation temperatures (65°C to 90°C) and durations (up to 6 hours).
- Sequencing of PCR amplicons was performed to identify fungal species.
Main Results:
- Increasing incubation temperature from 65°C to 90°C increased amplifiable DNA yield by up to 76-fold.
- Extended incubation up to 6 hours at 90°C further augmented DNA amplification.
- Improved fungal DNA amplification correlated with enhanced species identification through sequencing.
Conclusions:
- Optimizing thermal conditions during DNA extraction significantly enhances fungal DNA recovery from FFPE tissues.
- This improved molecular approach can help determine the etiology of IFI, even with challenging samples.
- Accurate etiological diagnosis through enhanced PCR and sequencing can guide antifungal therapy and improve patient management.
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