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Updated: Jul 26, 2025
DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses
Published on: March 26, 2011
The quality of DNA isolated from autopsy formalin-fixed and formalin-fixed paraffin-embedded tissues: study of 1662
Katarina Vitošević1,2, Danijela Todorović3, Živana Slović4,5
1Faculty of Medical Sciences, Department of Forensic Medicine, University of Kragujevac, Serbia, Kragujevac, Serbia. vitosevickatarina@gmail.com.
Background:
There are enormous formalin-fixed paraffin-embedded tissue archives and a constantly growing number of methods for molecular analyses but, the isolation of DNA from this tissue is still challenging due to the damaging effect of formalin on DNA. To determine the extent to which DNA purity, yield and integrity depend on the process of fixation in formalin, and to what extent on the process of tissue paraffin embedding, we compared the quality of DNA isolated from fixed tissues and DNA isolated from tissues embedded in paraffin blocks after fixation.
Methods And Results:
Heart, liver and brain tissues obtained from healthy people who suddenly died a violent death were fixed in 10% buffered formalin as well as in 4% unbuffered formalin for 6 h, 1-7 days (every 24 h), 10, 14, 28 days and 2 months. Additionally, the same tissues were fixed in 4% unbuffered formalin embedded in a paraffin block and stored from a few months to 30 years. The yield and purity of the DNA samples isolated from these tissues were measured using spectrophotometry. PCR amplification of the hTERT gene was performed to evaluate the degree of DNA fragmentation. Although the purity of the DNA isolated from almost all tissue samples was satisfactory, the DNA yields changed significantly. There was a decrease in successful PCR amplification of the hTERT gene in DNA samples isolated from tissue fixed in buffered and unbuffered formalin for up to 2 months from 100% to 8.3%. Archiving the tissue in paraffin blocks for up to 30 years also impacts the integrity of DNA, so there was a decrease in PCR amplification of the hTERT gene from 91% success to 3%.
Conclusion:
The largest decrease in DNA yield was observed after tissue formalin fixation after 14 days of fixation in buffered and unbuffered formalin. DNA integrity depends on the time of tissue formalin fixation, especially after 6 days for tissue fixed in unbuffered formalin, while for tissue fixed in buffered formalin the time is prolonged up to 28 days. The age of paraffin blocks also impacted DNA integrity, after 1 year and 16 years of archiving the paraffin blocks of tissues, there was a decrease in the success of PCR amplification.
Insights
Formalin fixation and paraffin embedding significantly impact DNA yield and integrity. DNA quality degrades over time with prolonged fixation and archival storage, affecting downstream molecular analyses.
Area of Science:
- Molecular Biology
- Genomics
- Biotechnology
Background:
- Vast archives of formalin-fixed paraffin-embedded (FFPE) tissues exist for molecular analyses.
- Formalin fixation poses challenges to DNA isolation due to DNA damage.
- Understanding fixation and embedding effects on DNA is crucial for FFPE sample utility.
Purpose of the Study:
- To assess DNA purity, yield, and integrity after formalin fixation and paraffin embedding.
- To compare DNA quality from fixed tissues versus tissues embedded post-fixation.
- To determine the impact of fixation duration and archival storage on DNA quality.
Main Methods:
- Human heart, liver, and brain tissues were fixed in buffered and unbuffered formalin for varying durations (6 hours to 2 months).
- Tissues were also embedded in paraffin blocks and stored for up to 30 years.
- DNA yield and purity were measured spectrophotometrically; DNA integrity was assessed via PCR amplification of the hTERT gene.
Main Results:
- DNA purity was generally satisfactory, but yields varied significantly.
- PCR amplification success decreased sharply with fixation duration, dropping to 8.3% after 2 months.
- Paraffin block storage up to 30 years also degraded DNA integrity, reducing PCR success to 3%.
Conclusions:
- Prolonged formalin fixation, especially beyond 14 days, significantly reduces DNA yield.
- DNA integrity is compromised by fixation duration, with unbuffered formalin showing effects after 6 days and buffered formalin after 28 days.
- Archival storage of FFPE tissues impacts DNA integrity, evidenced by reduced PCR amplification success over time.
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