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.
References
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Nuovo, G. J. (1997) PCR In Situ Hybridization, Protocols and Applications, 3rd ed., Lippinicott-Raven, New York.
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Nuovo, G.J. (2000). In Situ Localization of PCR-Amplified DNA and cDNA. In: Darby, I.A. (eds) In Situ Hybridization Protocols. Methods in Molecular Biology™, vol 123. Humana Press. https://doi.org/10.1385/1-59259-677-0:217
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DOI: https://doi.org/10.1385/1-59259-677-0:217
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