RAPID COMMUNICATION

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

Correspondence to: Clive R. Taylor, Dept. of Pathology, U. of Southern California School of Medicine, HMR 204, 2011 Zonal Avenue, Los Angeles, CA 90033. E-mail: taylor@pathfinder.hsc.usc.edu

During 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 heat-induced 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, paraffin-embedded 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. (J Histochem Cytochem 50:1005–1011, 2002)

Key Words: antigen retrieval, DNA extraction, formalin-fixed,, paraffin-embedded tissue, PCR

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				J. Guan, H.-L. Zhao, L. Baum, Y. Sui, L. He, H. Wong, F. M. M. Lai, P. C. Y. Tong, and J. C. N. Chan Apolipoprotein E polymorphism and expression in type 2 diabetic patients with nephropathy: clinicopathological correlation Nephrol. Dial. Transplant., June 1, 2009; 24(6): 1889 - 1895. [Abstract] [Full Text] [PDF]

				S. Yamashita, S. Takahashi, N. McDonell, N. Watanabe, T. Niwa, K. Hosoya, Y. Tsujino, T. Shirai, and T. Ushijima Methylation Silencing of Transforming Growth Factor-{beta} Receptor Type II in Rat Prostate Cancers Cancer Res., April 1, 2008; 68(7): 2112 - 2121. [Abstract] [Full Text] [PDF]

				A. S. Neimanis, H. N. Koopman, A. J. Westgate, K. Nielsen, and F. A. Leighton Evidence of Exposure to Brucella sp. in Harbor Porpoises (Phocoena phocoena) from the Bay of Fundy, Canada J. Wildl. Dis., April 1, 2008; 44(2): 480 - 485. [Abstract] [Full Text] [PDF]

				K. Hamatani, H. Eguchi, K. Takahashi, K. Koyama, M. Mukai, R. Ito, M. Taga, W. Yasui, and K. Nakachi Improved RT-PCR Amplification for Molecular Analyses with Long-term Preserved Formalin-fixed, Paraffin-embedded Tissue Specimens J. Histochem. Cytochem., July 1, 2006; 54(7): 773 - 780. [Abstract] [Full Text] [PDF]

				M. W.Y. Chan, S. H. Wei, P. Wen, Z. Wang, D. E. Matei, J. C. Liu, S. Liyanarachchi, R. Brown, K. P. Nephew, P. S. Yan, et al. Hypermethylation of 18S and 28S Ribosomal DNAs Predicts Progression-Free Survival in Patients with Ovarian Cancer Clin. Cancer Res., October 15, 2005; 11(20): 7376 - 7383. [Abstract] [Full Text] [PDF]

				B Thyagarajan, K E Anderson, F Kong, F R Selk, C F Lynch, and M D Gross New approaches for genotyping paraffin wax embedded breast tissue from patients with cancer: the Iowa women's health study J. Clin. Pathol., September 1, 2005; 58(9): 955 - 961. [Abstract] [Full Text] [PDF]

				H.-L. Zhao, P. C.Y. Tong, F. M.M. Lai, B. Tomlinson, and J. C.N. Chan Association of Glomerulopathy With the 5'-End Polymorphism of the Aldose Reductase Gene and Renal Insufficiency in Type 2 Diabetic Patients Diabetes, November 1, 2004; 53(11): 2984 - 2991. [Abstract] [Full Text] [PDF]

Guidelines | Subscriptions | About | exPRESS - Current - Archive | Business Information | Contact

The Journal of Histochemistry & Cytochemistry is owned, published, and licensed by The Histochemical Society © 2002