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In Situ Localization of PCR-Amplified DNA and cDNA

  • Gerard J. Nuovo
Part of the Methods in Molecular Biology™ book series (MIMB, volume 123)

Abstract

In situ hybridization is the only DNA- or RNA-based molecular biology based test that allows for the direct correlation of the results with the histologic and cytologic features of the sample. The DNA/RNA extraction that precedes filter hybridization (slot blot, hybrid capture, or Southern blot) and polymerase chain reaction (PCR) precludes this analysis. The relative sensitivities of the three different assays are presented in Table 1. It is evident that in situ hybridization is a relatively insensitive test. A reflection of this relative insensitivity is seen in occult or latent infection by a virus where the copy number is low. In such situations, the virus is rarely detected by in situ hybridization even though it was detected by either PCR or filter hybridization (1-6). This is not to say that the technique of in situ hybridization has remained static. The detection threshold of this assay has improved substantially in the last 10 years. Another point worth emphasizing about in situ hybridization is that one does not need to use radiolabeled probes (usually 35S or 3H) to maximize its sensitivity. Although true 10 years ago, recent and dramatic advances in nonisotopic labeling and, more importantly, detection systems has greatly enhanced the sensitivity when using such common labels as biotin and digoxigenin (7-12). Still, only the most aggressive salesman would claim (and incorrectly at that) that any given in situ system can routinely detect one DNA or RNA copy per cell. In my experience, this statement applies to the newer generation of posthybridization “signal amplification systems” (such as the cascade amplification system) which are not able to routinely detect one copy per cell.

Keywords

Polymerase Chain Reaction SiHa Cell Protease Digestion Saline Sodium Citrate Label Nucleotide 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    de Villiers, E. M., Schneider, A., and Miklaw, H. (1987) Human papillomavirus infections in women with and without abnormal cervical cytology. Lancet ii, 703–706.CrossRefGoogle Scholar
  2. 2.
    Nuovo, G. J. and Cottral, S. (1989) Occult infection of the uterine cervix by human papillomavirus in postmenopausal women. Am. J. Obstet. Gynecol. 160, 340–344.PubMedGoogle Scholar
  3. 3.
    Nuovo, G. J., Darfler, M. M., Impraim, C. C., and Bromley, S. E. (1991) Occurrence of multiple types of human papillomavirus in genital tract lesions: analysis by in situ hybridization and the polymerase chain reaction. Am. J. Pathol. 58, 518–523.Google Scholar
  4. 4.
    Nuovo, G. J., Hochman, H., Eliezri, Y. D., Comite, S., Lastarria, D., and Silvers, D. N. (1990) Detection of human papillomavirus DNA in penile lesions histologically negative for condylomata: analysis by in situ hybridization and the polymerase chain reaction. Am. J. Surg. Pathol. 14, 829–836.PubMedCrossRefGoogle Scholar
  5. 5.
    Nuovo, G. J. (1990) Human papillomavirus DNA in genital tract lesions histologically negative for condylomata, analysis by in situ, Southern blot hybridization and the polymerase chain reaction. Am. J. Surg. Pathol. 14, 643–651.PubMedCrossRefGoogle Scholar
  6. 6.
    Nuovo, G. J. (1989) A comparison of different methodologies (biotin based and 35S based.), for the detection of human papillomavirus DNA. Lab. Invest. 61, 471–476.PubMedGoogle Scholar
  7. 7.
    Crum, C. P., Nuovo, G. J., Friedman, D., and Silverstein, S. J. (1988) A comparison of biotin and isotope labeled ribonucleic acid probes for in situ detection of HPV 16 ribonucleic acid in genital precancers. Lab. Invest. 58, 354–359.PubMedGoogle Scholar
  8. 8.
    Nuovo, G. J. (1989) A comparison of slot blot, Southern blot and in situ hybridization analyses for human papillomavirus DNA in genital tract lesions. Obstet. Gynecol. 74, 673–677.PubMedGoogle Scholar
  9. 9.
    Ostrow, R. S., Manias, D. A., Clark, B. A., Okagaki, T., Twiggs, L. B., and Faras, A. J. (1987) Detection of human papillomavirus DNA in invasive carcinomas of the cervix by in situ hybridization. Cancer Res. 47, 649–653.PubMedGoogle Scholar
  10. 10.
    Walboomers, J. M. M., Melchers, W. J. G., and Mullink, H. (1988) Sensitivity of in situ detection with biotinylated probes of human papillomavirus type 16 DNA in frozen tissue sections of squamous cell carcinoma of the cervix. Am. J. Pathol. 131, 587–594.PubMedGoogle Scholar
  11. 11.
    Crum, C. P., Symbula, M., and Ward, B. E. (1989) Topography of early HPV 16 transcription in high-grade genital precancers. Am. J. Pathol. 134, 1183–1188.PubMedGoogle Scholar
  12. 12.
    Nagai, N., Nuovo, G. J., Friedman, D., and Crum, C. P. (1987) Detection of papillomavirus nucleic acids in genital precancers with the in situ hybridization technique, A review. Int. J. Gynecol. Pathol. 6, 366–379.PubMedCrossRefGoogle Scholar
  13. 13.
    Crum, C. P., Nuovo, G., Friedman, D., and Silverstein, S. J. (1988) Accumulation of RNA homologous to human papillomavirus type 16 open reading frames in genital precancers. J. Virol. 62, 84–90.PubMedGoogle Scholar
  14. 14.
    Crum, C. P., Nagai, N., Levine, R. U., and Silverstein, S. J. (1986) In situ hybridization analysis of human papillomavirus 16 DNA sequences in early cervical neoplasia. Am. J. Pathol. 123, 174–182.PubMedGoogle Scholar
  15. 15.
    Nuovo, G. J. and Silverstein, S. J. (1988) Comparison of formalin, buffered formalin, and Bouin’s fixation on the detection of human papillomavirus DNA from genital lesions. Lab. Invest. 59, 720–724.PubMedGoogle Scholar
  16. 16.
    Nuovo, G. J., Friedman, D., Silverstein, S. J., and Crum, C. P. (1987) Transcription of human papillomavirus type 16 in genital precancers. Cancer Cells. 5, 337–343.Google Scholar
  17. 17.
    Lebargy, F., Bulle, F., Siegrist, S., Guellaen, G., and Bernaudin, J. (1990) Localization by in situ hybridization of γ glutamyl transpeptidase mRNA in the rat kidney using 35S-labeled RNA probes. Lab. Invest. 62, 731–735.PubMedGoogle Scholar
  18. 18.
    McAllister, H. A. and Rock, D. L. (1985) Comparative usefulness of tissue fixatives for in situ viral nucleic acid hybridization. J. Histochem. Cytochem. 33, 1026–1032.PubMedGoogle Scholar
  19. 19.
    Greer, C. E., Peterson, S. L., Kiviat, N. B. and Manos, M. M. (1991) PCR amplification from paraffin-embedded tissues: effects of fixative and fixative times. Am. J. Clin. Pathol. 95, 117–124.PubMedGoogle Scholar
  20. 20.
    Nuovo, G. J. (1989) Buffered formalin is the superior fixative for the detection of human papillomavirus DNA by in situ hybridization analysis. Am. J. Pathol. 134, 837–842.PubMedGoogle Scholar
  21. 21.
    Tournier, I., Bernuau, D., Poliard, A., Schoevaert, D., and Feldmann, G. (1987) Detection of albumin mRNAs in rat liver by in situ hybridization, usefulness of paraffin embedding and comparison of various fixation procedures. J. Histochem. Cytochem. 35, 453–459.PubMedGoogle Scholar
  22. 22.
    Nuovo, G. J. (1991) Comparison of Bouin’s solution and buffered formalin fixation on the detection rate by in situ hybridization of human papillomavirus DNA in genital tract lesions. J. Histotech. 14, 13–18.Google Scholar
  23. 23.
    Nuovo, G. J., Gallery, F., Hom, R., MacConnell, P., and Bloch, W. (1993) Importance of different variables for optimizing in situ detection of PCR-amplified DNA. PCR. Method Appl. 2, 305–312.Google Scholar
  24. 24.
    Goelz, S. E., Hamilton, S. R., and Vogelstein, B. (1985) Purification of DNA from formaldehyde fixed and paraffin-embedded tissue. Biochem. Biophys. Res. Commun. 130, 118–124.PubMedCrossRefGoogle Scholar
  25. 25.
    Bromley, S. E., Darfler, M. M., Hammer, M. L., Jones-Trower, A., Primus, M. A., and Kreider, J. W. (1990) In situ hybridization to human papillomavirus DNA in fixed tissue samples, Comparison of detection methods, in Papillomaviruses (Crum, C. P., ed.), Wiley-Liss, New York, 46–53.Google Scholar
  26. 26.
    Nuovo, G. J., MacConnell, P. and Becker, J. (1992) Rapid in situ detection of PCR-amplified HIV-1 DNA. Diagn. Mol. Pathol. 1, 98–102.PubMedGoogle Scholar
  27. 27.
    Nuovo, G. J., Gallery, F., and MacConnell, P. (1992) Analysis of the distribution pattern of PCR-amplified HPV 6 DNA in vulvar warts by in situ hybridization. Mod. Pathol. 5, 444–448.PubMedGoogle Scholar
  28. 28.
    Nuovo, G. J., Becker, J., MacConnell, P., Comite, S., and Hochman, H. (1992) Histological distribution of PCR-amplified HPV 6 and 11 DNA in penile lesions. Am. J. Surg. Pathol. 16, 269–275.PubMedCrossRefGoogle Scholar
  29. 29.
    Nuovo, G. J., MacConnell, P., Forde, A., and Delvenne, P. (1991) Detection of human papillomavirus DNA in formalin fixed tissues by in situ hybridization after amplification by PCR. Am. J. Pathol. 139, 847–854.PubMedGoogle Scholar
  30. 30.
    Nuovo, G. J., Gallery, F., MacConnell, P., Becker, J., and Bloch, W. (1991) An improved technique for the detection of DNA by in situ hybridization after PCR-amplification. Am. J. Pathol. 139, 1239–1244.PubMedGoogle Scholar
  31. 31.
    Chou, Q., Russell, M., Birch, D. E., Raymond, J., and Bloch, W. (1992) Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications. Nucleic Acids Res. 20, 1717–1723.PubMedCrossRefGoogle Scholar
  32. 32.
    Nuovo, G. J., Gorgone, G., MacConnell, P., and Goravic, P. (1992) In situ localization of human and viral cDNAs after PCR-amplification. PCR Method Appl. 2, 117–123.Google Scholar
  33. 33.
    Nuovo, G. J. (1997) PCR In Situ Hybridization, Protocols and Applications, 3rd ed., Lippinicott-Raven, New York.Google Scholar

Copyright information

© Humana Press Inc. 2000

Authors and Affiliations

  • Gerard J. Nuovo
    • 1
  1. 1.MGN Medical Research LaboratoriesSetauket

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