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International Journal of Experimental Pathology logoLink to International Journal of Experimental Pathology
. 1993 Aug;74(4):333–337.

DNA amplification by polymerase chain reaction from brain tissues embedded in paraffin.

K Gall 1, J Pavelić 1, D Jadro-Santel 1, M Poljak 1, K Pavelić 1
PMCID: PMC2001845  PMID: 8398805

Abstract

A method which enables analysis of DNA from archival paraffin embedded normal and malignant brain tissue is described. The demonstration of a 317-bp long beta-actin DNA sequence by the polymerase chain reaction (PCR) was used to identify which fixation procedure, deparaffinization time and DNA extraction procedure would give the best results. Tissue specimens 1-39 years old were included in the experiments. Specimens fixed in either 10% formalin, Carnoy's or AMeX fixative were found to be best suited for subsequent analysis by PCR. Paraformaldehyde and acetone compromised amplification efficiency, while Bouin's fixed tissue gave uniformly negative results. Regardless of fixative used, PCR reaction had to be run through at least 40 cycles. Prolonged deparaffinization time and phenol/chloroform extraction of DNA did not influence DNA quality as a template for PCR reaction. Formalin fixed brain tumours can be successfully used for DNA/PCR analysis even if they are up to 39 years old.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ben-Ezra J., Johnson D. A., Rossi J., Cook N., Wu A. Effect of fixation on the amplification of nucleic acids from paraffin-embedded material by the polymerase chain reaction. J Histochem Cytochem. 1991 Mar;39(3):351–354. doi: 10.1177/39.3.1704393. [DOI] [PubMed] [Google Scholar]
  2. Coates P. J., d'Ardenne A. J., Khan G., Kangro H. O., Slavin G. Simplified procedures for applying the polymerase chain reaction to routinely fixed paraffin wax sections. J Clin Pathol. 1991 Feb;44(2):115–118. doi: 10.1136/jcp.44.2.115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Dubeau L., Chandler L. A., Gralow J. R., Nichols P. W., Jones P. A. Southern blot analysis of DNA extracted from formalin-fixed pathology specimens. Cancer Res. 1986 Jun;46(6):2964–2969. [PubMed] [Google Scholar]
  4. Goelz S. E., Hamilton S. R., Vogelstein B. Purification of DNA from formaldehyde fixed and paraffin embedded human tissue. Biochem Biophys Res Commun. 1985 Jul 16;130(1):118–126. doi: 10.1016/0006-291x(85)90390-0. [DOI] [PubMed] [Google Scholar]
  5. Greer C. E., Peterson S. L., Kiviat N. B., Manos M. M. PCR amplification from paraffin-embedded tissues. Effects of fixative and fixation time. Am J Clin Pathol. 1991 Feb;95(2):117–124. doi: 10.1093/ajcp/95.2.117. [DOI] [PubMed] [Google Scholar]
  6. Impraim C. C., Saiki R. K., Erlich H. A., Teplitz R. L. Analysis of DNA extracted from formalin-fixed, paraffin-embedded tissues by enzymatic amplification and hybridization with sequence-specific oligonucleotides. Biochem Biophys Res Commun. 1987 Feb 13;142(3):710–716. doi: 10.1016/0006-291x(87)91472-0. [DOI] [PubMed] [Google Scholar]
  7. Jackson D. P., Lewis F. A., Taylor G. R., Boylston A. W., Quirke P. Tissue extraction of DNA and RNA and analysis by the polymerase chain reaction. J Clin Pathol. 1990 Jun;43(6):499–504. doi: 10.1136/jcp.43.6.499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Jackson D. P., Quirke P., Lewis F., Boylston A. W., Sloan J. M., Robertson D., Taylor G. R. Detection of measles virus RNA in paraffin-embedded tissue. Lancet. 1989 Jun 17;1(8651):1391–1391. doi: 10.1016/s0140-6736(89)92837-7. [DOI] [PubMed] [Google Scholar]
  9. Nuovo G. J., Richart R. M. Buffered formalin is the superior fixative for the detection of HPV DNA by in situ hybridization analysis. Am J Pathol. 1989 Apr;134(4):837–842. [PMC free article] [PubMed] [Google Scholar]
  10. Pavelic K., Pavelic Z. P., Preisler H. D. c-myc detection in bone marrow biopsies. Leuk Res. 1991;15(11):1075–1078. doi: 10.1016/0145-2126(91)90114-9. [DOI] [PubMed] [Google Scholar]
  11. Rogers B. B., Alpert L. C., Hine E. A., Buffone G. J. Analysis of DNA in fresh and fixed tissue by the polymerase chain reaction. Am J Pathol. 1990 Mar;136(3):541–548. [PMC free article] [PubMed] [Google Scholar]
  12. Schoenberg B. S., Christine B. W., Whisnant J. P. The descriptive epidemiology of primary intracranial neoplasms: the Connecticut experience. Am J Epidemiol. 1976 Nov;104(5):499–510. doi: 10.1093/oxfordjournals.aje.a112322. [DOI] [PubMed] [Google Scholar]
  13. Seizinger B. R., Rouleau G., Ozelius L. J., Lane A. H., St George-Hyslop P., Huson S., Gusella J. F., Martuza R. L. Common pathogenetic mechanism for three tumor types in bilateral acoustic neurofibromatosis. Science. 1987 Apr 17;236(4799):317–319. doi: 10.1126/science.3105060. [DOI] [PubMed] [Google Scholar]
  14. Seizinger B. R., de la Monte S., Atkins L., Gusella J. F., Martuza R. L. Molecular genetic approach to human meningioma: loss of genes on chromosome 22. Proc Natl Acad Sci U S A. 1987 Aug;84(15):5419–5423. doi: 10.1073/pnas.84.15.5419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Shibata D. K., Arnheim N., Martin W. J. Detection of human papilloma virus in paraffin-embedded tissue using the polymerase chain reaction. J Exp Med. 1988 Jan 1;167(1):225–230. doi: 10.1084/jem.167.1.225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Shibata D., Martin W. J., Arnheim N. Analysis of DNA sequences in forty-year-old paraffin-embedded thin-tissue sections: a bridge between molecular biology and classical histology. Cancer Res. 1988 Aug 15;48(16):4564–4566. [PubMed] [Google Scholar]
  17. Tokuda Y., Nakamura T., Satonaka K., Maeda S., Doi K., Baba S., Sugiyama T. Fundamental study on the mechanism of DNA degradation in tissues fixed in formaldehyde. J Clin Pathol. 1990 Sep;43(9):748–751. doi: 10.1136/jcp.43.9.748. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Wan J. H., Trainor K. J., Brisco M. J., Morley A. A. Monoclonality in B cell lymphoma detected in paraffin wax embedded sections using the polymerase chain reaction. J Clin Pathol. 1990 Nov;43(11):888–890. doi: 10.1136/jcp.43.11.888. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. von Weizsäcker F., Labeit S., Koch H. K., Oehlert W., Gerok W., Blum H. E. A simple and rapid method for the detection of RNA in formalin-fixed, paraffin-embedded tissues by PCR amplification. Biochem Biophys Res Commun. 1991 Jan 15;174(1):176–180. doi: 10.1016/0006-291x(91)90502-x. [DOI] [PubMed] [Google Scholar]

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