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      Comparison of Methods for the Extraction of DNA from Formalin-Fixed, Paraffin-Embedded Archival Tissues

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          Abstract

          Aim: Discussing a protocol involving xylene-ethanol deparaffinization on slides followed by a kit-based extraction that allows for the extraction of high quality DNA from FFPE tissues.

          Methods: DNA was extracted from the FFPE tissues of 16 randomly selected blocks. Methods involving deparaffinization on slides or tubes, enzyme digestion overnight or for 72 hours and isolation using phenol chloroform method or a silica-based commercial kit were compared in terms of yields, concentrations and the amplifiability.

          Results: The highest yield of DNA was produced from the samples that were deparaffinized on slides, digested for 72 hours and isolated with a commercial kit. Samples isolated with the phenol-chloroform method produced DNA of lower purity than the samples that were purified with kit. The samples isolated with the commercial kit resulted in better PCR amplification.

          Conclusion: Silica-based commercial kits and deparaffinized on slides should be considered for DNA extraction from FFPE.

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          A high frequency of sequence alterations is due to formalin fixation of archival specimens.

          C. Williams, F Ponten, C Moberg … (1999)
          Genomic analysis of archival tissues fixed in formalin is of fundamental importance in biomedical research, and numerous studies have used such material. Although the possibility of polymerase chain reaction (PCR)-introduced artifacts is known, the use of direct sequencing has been thought to overcome such problems. Here we report the results from a controlled study, performed in parallel on frozen and formalin-fixed material, where a high frequency of nonreproducible sequence alterations was detected with the use of formalin-fixed tissues. Defined numbers of well-characterized tumor cells were amplified and analyzed by direct DNA sequencing. No nonreproducible sequence alterations were found in frozen tissues. In formalin-fixed material up to one mutation artifact per 500 bases was recorded. The chance of such artificial mutations in formalin-fixed material was inversely correlated with the number of cells used in the PCR-the fewer cells, the more artifacts. A total of 28 artificial mutations were recorded, of which 27 were C-T or G-A transitions. Through confirmational sequencing of independent amplification products artifacts can be distinguished from true mutations. However, because this problem was not acknowledged earlier, the presence of artifacts may have profoundly influenced previously reported mutations in formalin-fixed material, including those inserted into mutation databases.
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            Purification of DNA from formaldehyde fixed and paraffin embedded human tissue.

            Ian Hamilton, B Vogelstein, S E Goelz (1985)
            The ability to isolate DNA from preserved human tissues would provide numerous experimental opportunities. In this report it is shown that DNA can be extracted from tissues prepared for routine histopathological examination (i.e., fixed with formaldehyde and embedded in paraffin). Although the extracted DNA is not intact, it is double stranded, cleavable with restriction endonucleases, and suitable for a variety of standard techniques used in molecular biology.
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              Formaldehyde-mediated DNA-protein crosslinking: a probe for in vivo chromatin structures.

              M Solomon, A Varshavsky (1985)
              Formaldehyde (HCHO) produces DNA-protein crosslinks both in vitro and in vivo. Simian virus 40 (SV40) chromosomes that have been fixed by prolonged incubation with HCHO either in vitro or in vivo (within SV40-infected cells) can be converted to nearly protein-free DNA by limit-digestion with Pronase in the presence of NaDodSO4. The remaining Pronase-resistant DNA-peptide adducts retard the DNA upon gel electrophoresis, allowing resolution of free and crosslink-containing DNA. Though efficiently crosslinking histones to DNA within nucleosomes both in vitro and in vivo, HCHO does not crosslink either purified lac repressor to lac operator-containing DNA or an (A + T)-DNA-binding protein (alpha-protein) to its cognate DNA in vitro. Furthermore, a protein that does not bind to DNA, such as serum albumin, is not crosslinked to DNA by HCHO even at extremely high protein concentrations. These properties of HCHO as a DNA-protein crosslinker are used to probe the distribution of nucleosomes in vivo. We show that there are no HCHO-crosslinkable DNA-protein contacts in a subset of SV40 chromosomes in vivo within a 325-base-pair stretch that spans the "exposed" (nuclease-hypersensitive) region of the SV40 chromosome. This replication origin-proximal region has been found previously to lack nucleosomes in a subset of isolated SV40 chromosomes. We discuss other applications of the HCHO technique, including the possibility of obtaining base-resolution in vivo nucleosome "footprints."
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                Author and article information

                Journal
                Journal ID (nlm-ta): Int J Med Sci
                Journal ID (iso-abbrev): Int J Med Sci
                Journal ID (publisher-id): ijms
                Title: International Journal of Medical Sciences
                Publisher: Ivyspring International Publisher (Sydney )
                ISSN (Electronic): 1449-1907
                Publication date Collection: 2014
                Publication date (Electronic): 27 March 2014
                Volume: 11
                Issue: 5
                Pages: 494-499
                Affiliations
                1. Department of Oral Pathology, Faculty of Dentistry, Gazi University, Ankara, Turkey;
                2. Pharmacoeconomy and Pharmacoepidemiology Research Center (PEPIRC), Yeditepe University, Istanbul, Turkey.
                Author notes
                ✉ Corresponding author: Dr. Burcu Sengüven, Department of Oral Pathology, Faculty of Dentistry, Gazi University. ANKARA 06510, TURKEY. Telephone: +90 312 2034384 Fax: +90312 2239226 E-mail: senguvenb@ 123456yahoo.com .

                Competing Interests: The authors have declared that no competing interest exists.

                Article
                Publisher ID: ijmsv11p0494
                DOI: 10.7150/ijms.8842
                PMC ID: 3970103
                PubMed ID: 24688314
                SO-VID: b1a5eb53-1ab2-43ad-a21c-295ffcc91299
                Copyright © © Ivyspring International Publisher. This is an open-access article distributed under the terms of the Creative Commons License (http://creativecommons.org/licenses/by-nc-nd/3.0/). Reproduction is permitted for personal, noncommercial use, provided that the article is in whole, unmodified, and properly cited.
                History
                Date received : 15 February 2014
                Date accepted : 13 March 2014
                Categories
                Subject: Research Paper

                ScienceOpen disciplines: Medicine
                Keywords: dna extraction,formalin fixed paraffin-embedded tissues.
                Data availability:
                ScienceOpen disciplines: Medicine
                Keywords: dna extraction, formalin fixed paraffin-embedded tissues.

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