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Updated: Aug 10, 2026
qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue
Published on: July 10, 2014
Sensitivity of the polymerase chain reaction for detecting human T-cell leukemia virus type I sequences in
1Department of Neurological Sciences, Tohoku University School of Medicine, Sendai, Japan.
Abstract:
Recently, the application of the polymerase chain reaction (PCR) to formalin-fixed paraffin-embedded tissue has been reported. But formalin, especially unbuffered formalin, is known to break DNA into small fragments. DNA extracted from MT-2 cells fixed in unbuffered formalin for various periods of time were subjected to the PCR and the effect of unbuffered formalin fixation on the ability of the PCR to detect exogenous sequences; i.e., human T-cell leukemia virus type I (HTLV-I) proviral DNA, was examined. The sensitivity of the PCR decreased as a function of both the duration of fixation and the length of the expected DNA products. When the expected length of the PCR product was about 200 bp, a slight decrease in the sensitivity was observed after 4-day fixation. When it was about 300 bp, a similar decrease was observed following 4-h fixation. In the case of a 500 bp product, the sensitivity began to decrease after 30-min fixation and a 100-fold decrease was observed after 10-day fixation. A decrease was not observed, however, with a 100 bp product. The appropriate design of primers, especially with regard to the length of the amplified product, is essential to keep the sensitivity of the PCR, particularly when the target tissues have been fixed in unbuffered formalin.
Insights
Formalin fixation degrades DNA, impacting polymerase chain reaction (PCR) sensitivity. Shorter PCR products and shorter fixation times are essential for detecting target DNA in formalin-fixed tissues.
Area of Science:
- Molecular Biology
- Genetics
- Histology
Background:
- Formalin-fixed paraffin-embedded (FFPE) tissues are crucial for molecular studies.
- Formalin, particularly unbuffered solutions, can fragment DNA, potentially hindering downstream analyses like PCR.
Purpose of the Study:
- To investigate the impact of unbuffered formalin fixation duration on the sensitivity of PCR.
- To evaluate how the length of DNA targets affects PCR detection in FFPE tissues.
Main Methods:
- DNA was extracted from MT-2 cells fixed in unbuffered formalin for varying durations.
- Extracted DNA was subjected to PCR to detect exogenous human T-cell leukemia virus type I (HTLV-I) proviral DNA.
- PCR sensitivity was assessed based on the length of the amplified DNA product.
Main Results:
- PCR sensitivity decreased with increased formalin fixation duration and longer DNA product lengths.
- Detection of a 500 bp product showed a 100-fold decrease in sensitivity after 10-day fixation.
- A 100 bp product remained detectable even after extended fixation periods, indicating minimal sensitivity loss.
Conclusions:
- Primer design, specifically targeting shorter DNA fragments, is critical for maintaining PCR sensitivity in unbuffered formalin-fixed tissues.
- Optimizing fixation protocols and PCR strategies is essential for reliable molecular analysis of FFPE samples.
