DNA Extraction from Archival Formalin-fixed, Paraffin-embedded Tissue Sections Based on the Antigen Retrieval Principle: Heating Under the Influence of pH

Abstract

S U M M A R YDuring the course of diagnostic surgical pathology, pathologists have established a large collection of formalin-fixed, paraffin-embedded tissues that form invaluable resources for translational studies of cancer and a variety of other diseases. Accessibility of macromolecules in the fixed tissue specimens is a critical issue as exemplified by heatinduced antigen retrieval (AR) immunohistochemical (IHC) staining. On the basis of observations that heating may also enhance in situ hybridization (ISH) and the similarity of formalin-induced chemical modifications that occur in protein and in DNA, we designed a study to examine the efficiency of DNA extraction from archival formalin-fixed, paraffinembedded tissues using an adaptation of the basic principles of the AR technique, i.e., heating the tissue under the influence of different pH values. Archival paraffin blocks of lymph nodes, tonsil, and colon were randomly selected. Each paraffin block was prepared in 34 microtubes. For each paraffin block, one tube was used as a control sample, using a non-heating DNA extraction protocol. The other 33 tubes were tested using a heating protocol under 11 variable pH values (pH 2 to 12) under three different heating conditions (80, 100, and 120C). Evaluation of the results of DNA extraction was carried out by measuring yields by photometry and PCR amplification, as well as kinetic thermocycling (KTC)-PCR methods. In general, lower pH (acid) solutions gave inferior results to solutions at higher pH (alkaline). Heating tissues at a higher temperature and at pH 6-9 gave higher yields of DNA. There appeared to be a peak in terms of highest efficiency of extracted DNA at around pH 9. The average ratios 260:280 of extracted DNA also showed better values for samples heated at 120C. PCR products of three primers showed satisfactory results for DNA extracted from archival paraffin-embedded tissues by heating protocols at pH 6-12, with results that were comparable to the control sample subjected to the standard non-heating, enzymatic DNA extraction method. This study is the first to document the use of heating at an alkaline pH for DNA extraction from archival formalin-fixed, paraffin-embedded tissues, a recommendation based on the principles of AR for protein IHC. These findings may lead to a more effective protocol for DNA extraction from archival paraffin-embedded tissues and may also provide enhanced understanding of changes that occur during formalin-induced modification of nucleic acids.

R462Q Mutation in Prostate Cancer Specimens

“…The allelic frequency for R462Q in cancerous and non-cancerous patients was determined to be 0.108 and 0.064, respectively. It is the same as the results of a study by Shea et al (12) and lower in comparison to other prostate cancer studies (the allel frequency of R462Q, 0.25 and 0.38) (9,10,13,14). Prostate cancer was not found to be significantly associated with the R462Q polymorphism of RNASEL (P > 5% Fisher's exact test) ( Table 2).…”

“…As found in the epidemiological studies, there were diverse associations between Arg462Gln polymorphism and prostate cancer in sporadic and familial prostate cases (3,5,(8)(9)(10). Therefore, in this study we evaluated the association between this polymorphism and prostate cancer in a cross-sectional study.…”

Efficient DNA Extraction for HPV Genotyping in Formalin-Fixed, Paraffin-Embedded Tissues

“…Such a method applies a less efficient lower heat treatment 3 and relies on the availability of an automated VERSANT kPCR system (Siemens, Erlangen, Germany) with silica-coated iron oxide beads. Our protocol can likely be adapted to most automated methods for DNA extraction.…”

“…1 DNA cross-links can be reversed to some extent by heat, and various temperature and incubation times have been proposed. [2][3][4] The majority of protocols for nucleic acid extraction from FFPE tissues traditionally incorporate a pretreatment with xylene and ethanol to physically remove the paraffin wax. 5 The disadvantages of this procedure include labor intensity, chemical toxicity, and the risk of accidentally removing small tissue fragments during the process.…”

“…We found that the current published protocols are either too laborious or do not reliably produce DNA in sufficient quantity for this objective. We therefore combined a 120°C heat treatment, as suggested by Shi et al, 3 with a commercially available DNA isolation kit. The resulting extracts were compared with DNA isolated in accordance with the manufacturer's protocol and were evaluated with regards to HPV typing.…”