DNA extraction from archival formalin-fixed, paraffin-embedded tissues: heat-induced retrieval in alkaline solution

Abstract

Based on the antigen retrieval principle, our previous study has demonstrated that heating archival formalin-fixed, paraffin-embedded (FFPE) tissues at a higher temperature and at higher pH value of the retrieval solution may achieve higher efficiency of extracted DNA, when compared to the traditional enzyme digestion method. Along this line of heat-induced retrieval, this further study is focused on development of a simpler and more effective heat-induced DNA retrieval technique by testing various retrieval solutions. Three major experiments using a high temperature heating method to extract DNA from FFPE human lymphoid and other tissue sections were performed to compare: (1) different concentrations of alkaline solution (NaOH or KOH, pH 11.5-12) versus Britton and Robinson type of buffer solution (BR buffer) of pH 12 that was the only retrieval solution tested in our previous study; (2) several chemical solutions (SDS, Tween 20, and GITC of various concentrations) versus BR buffer or alkaline solution; and (3) alkaline solution mixed with chemicals versus BR buffer or single alkaline solution. Efficiency of DNA extraction was evaluated by measuring yields using spectrophotometry, electrophoretic pattern, semiquantitation of tissue dissolution, PCR amplification, and kinetic thermocycling-PCR methods. Results showed that boiling tissue sections in 0.1 M NaOH or KOH or its complex retrieval solutions produced higher yields and better quality of DNA compared to BR buffer or chemical solutions alone. The conclusion was that boiling FFPE tissue sections in 0.1 M alkaline solution is a simpler and more effective heat-induced retrieval protocol for DNA extraction. Combination with some chemicals (detergents) may further significantly improve efficiency of the heat-induced retrieval technique.

Do Antigen Retrieval Techniques Improve DNA Yield from Formalin Fixed Paraffin Embedded Tissue?

“…The use of these solutions together with the high temperature of pressure cooking may enable the reversal of the crosslinking effect of formalin in FFPE tissues. Our results correlate with Shi and colleagues with respect to pH values of the solutions employed, as both studies detected an increase in DNA yield with higher pH values (6-9) [6,15]. The increased DNA concentration, that was accompanied by an increase in the 260/280 OD ratio with our pressure cooked samples indicates that this technique is an improvement from the standard protocol previously used.…”

ACMG technical standards and guidelines for genetic testing for inherited colorectal cancer (Lynch syndrome, familial adenomatous polyposis, and MYH-associated polyposis)

“…The digested sections (DNA lysates) can be directly used for MSI testing, or DNA can be extracted using standard DNA extraction methods using glycogen as a carrier to enhance yield. 58 2.13.4 BRAF gene p.V600E missense mutation The p.V600E mutation can be detected using a targeted mutation detection technique, such as restriction enzyme digestion, allele-specific primer extension, or real-time PCR. Refer to ACMG Standards and Guidelines for Clinical Genetics Laboratories, Section G8.7, for technical details.…”

An efficient protocol for genomic DNA extraction from formalin-fixed paraffin-embedded tissues

“…However, a growing number of authors do not employ specific protocols to remove the paraffin (e.g. Stanta and Schneider, 1991;Wu et al,2002;Shi et al, 2004).…”