Abstract
This study investigated the use of pressure cooking with different retrieval solutions at variable pHs to determine the optimum protocol for the extraction of high quality DNA from formalin fixed paraffin embedded (FFPE) tissues. Further, the oxidation effect on the archived FFPE tissues in relation to the age of the tissue blocks was also explored. The main aim of the study was to investigate this technique on 11 gastric cancers. The design of our study was based on the principles of the antigen retrieval technique using a conventional pressure cooker to improve the quality of DNA extracted from FFPE tissues. In addition to the gastric cancers, lymphoma, breast, prostatic, and colorectal carcinomas were used to eliminate tissue bias. Statistical correlation was done using the paired t-test and Benjamini-Hochberg test. Our findings show that high DNA concentrations were obtained using the different retrieval solutions with pressure cooking, compared to the control samples that were not subjected to this procedure. The mean DNA concentration increased with all the solutions tested but DNA yield was significantly higher in 3 of the 4 retrieval solutions used. The use of antigen retrieval solutions at high pressure and temperature provided by pressure cooking may enable the reversal of the crosslinking effect of formalin in FFPE tissues. Further, the observation that the DNA extracted from recently processed blocks was of a superior yield as opposed to older tissue blocks, confirms that oxidation does have a deteriorating effect on DNA.
Highlights
DNA extracted from formalin fixed paraffin embedded (FFPE) tissues is often significantly degraded and extraction of good quality DNA remains a challenge
There are a number of factors that contribute to DNA degradation, which include: type of fixative, duration of fixation, duration of tissue hypoxia, permeability of fixative and the duration of storage of the paraffin wax blocks [2]
In view of the above it is very likely that antigen retrieval solutions used in immunohistochemistry would enhance the quality of DNA extracted from FFPE tissues
Summary
DNA extracted from formalin fixed paraffin embedded (FFPE) tissues is often significantly degraded and extraction of good quality DNA remains a challenge. Whilst formalin fixation is the method of choice in most diagnostic histopathological laboratories, this form of tissue fixation results in varying degrees of DNA degradation [1,2,3,4]. Studies have shown that the chemical reactions between formalin and DNA are similar to that of formalin and proteins [4,5,6]. The chemical mechanism by which formalin induces DNA denaturation has been well documented [2,4,7]. The following are the dominant interactions in formalin fixation that support the hypothesis of our study: yyAn initial reaction is the rapid and reversible hydroxymethylation of the imino and amino groups of nucleic acid bases to form a hydroxymethyl (methylol) group (-CH2OH) [2,7]
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