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The use of optimal cutting temperature compound can inhibit amplification by polymerase chain reaction
1Department of Pathology, Royal Perth Hospital, Western Australia, Australia.
Abstract:
Optimal cutting temperature (OCT) is a widely used embedding medium for tissues for histopathologic analysis. This investigation examined the effects that OCT storage can have upon the ability to perform subsequent molecular biological analyses. Tumor material was dissected into small pieces and stored at approximately 20 degrees C both with and without OCT. DNA and RNA were then extracted from the tissue fragments and analyzed by the polymerase chain reaction (PCR), using primer sets designed to amplify a range of product sizes, and also by reverse transcriptase-PCR (RT-PCR). The storage of pathological specimens in OCT compound was found to affect significantly and irreversibly the ability to amplify DNA in the PCR, particularly as the size of the amplified fragment increased. This effect appeared to occur as a result of greater degradation of DNA extracted from tissue embedded in OCT compared to DNA extracted from tissue stored without OCT. RNA quality appeared unaffected, which may be because of the extraction protocol employed. Our results suggest that OCT-embedded frozen-tissue samples may be used for RNA isolation for subsequent RT-PCR and for the in vitro amplification of DNA targets of approximately < 300 base pairs only. We strongly advise against the routine storage of any tissue biopsy material in OCT if molecular analyses may be required.
Insights
Storing tissue in Optimal Cutting Temperature (OCT) medium significantly degrades DNA, hindering PCR amplification, especially for larger fragments. RNA remains largely unaffected, but OCT embedding is not recommended for molecular analyses.
Area of Science:
- Histopathology
- Molecular Biology
- Biochemistry
Background:
- Optimal Cutting Temperature (OCT) is a common embedding medium for histopathology.
- Its impact on subsequent molecular analyses is not fully understood.
Purpose of the Study:
- To investigate the effects of OCT storage on DNA and RNA integrity for molecular analyses.
- To determine the suitability of OCT-embedded tissues for PCR and RT-PCR.
Main Methods:
- Tumor tissues were stored with and without OCT at 20°C.
- DNA and RNA were extracted and analyzed using PCR and RT-PCR with varying product sizes.
Main Results:
- OCT storage significantly and irreversibly impaired DNA amplification in PCR, particularly for larger fragments (>300 bp).
- Increased DNA degradation was observed in OCT-embedded tissues compared to non-embedded tissues.
- RNA quality remained unaffected, potentially due to the extraction protocol.
Conclusions:
- OCT embedding negatively impacts DNA integrity, limiting its use for PCR amplification of larger targets.
- OCT-embedded tissues are suitable for RNA isolation for RT-PCR and for amplifying small DNA targets (<300 bp).
- Routine storage of tissue in OCT is not recommended when molecular analyses are anticipated.