Abstract
In contrast to the immediate and enormous impact that solution-phase polymerase chain reaction (PCR) had in molecular biology, morphologists have regarded the development of in situ PCR with cautious expectancy (1,2). The notion of employing a PCR-based amplification step to increase the sensitivity of in situ hybridization (ISH) apparently came to several laboratories independently in the late 1980s (3–6). The result has been a relatively small number of publications detailing at once some successes, certain problems and limitations of the technique, and a variety of different technical approaches to combining the technologies of ISH and PCR. As we approach the end of the 1990s, it seems likely that the relative importance of the variables identified will be agreed on and that some consensus concerning optimization of the multiple steps involved will emerge, paving the way for standardized protocols that may serve to establish in situ PCR in diagnostic laboratories.
References
Murray, G. (1993) In situ PCR (editorial). J. Pathol. 169, 187,188.
Hofler, H. (1993) In situ polymerase chain reaction: toy or tool? (editorial). Histochemistry 99, 103,104.
Haase, A. T., Retzel, E. F., and Staskus, K. A. (1990) Amplification and detection of lentiviral DNA inside cells. Proc. Natl. Acad. Sci USA 87, 4971–4975.
Spann, W., Pachmann, K., Zabnienska, H., Pielmeier, A., and Emmerich, B. (1991) In situ amplification of single copy gene segments in individual cells by the polymerase chain reaction. Infection 19, 242–244.
Ray, R., Komminoth, P., Machado, M., and Wolfe, H. J. (1991) Combined polymerase chain reaction and in-situ hybridization for the detection of single copy genes and viral genomic sequences in intact cells. Modern Pathol. 4, 124A.
Nuovo, G., MacConnell, P., Forde, A., and Delvenne, P. (1991) Detection of human papillomavirus DNA in formalin-fixed tissues by in situ hybridization after amplification by polymerase chain reaction. Am. J. Pathol. 139, 847–854.
Bagasra, O., Seshamma, T., and Pomerantz, R. (1993) Polymerase chain reaction in situ: intracellular amplification and detection of HIV-1 proviral DNA and other specific genes. J. Immunol. Methods 158, 131–145.
Teo, I. A. and Shaunak, S. (1995) Polymerase chain reaction in situ: an appraisal of an emerging technique. Histochem. J. 27, 647–659.
Nuovo, G. (1992) PCR In Situ Hybridization. Raven, NY.
Embleton, M., Gorochov, G., Jones, P., and Winter, G. (1992) In-cell PCR from mRNA: amplifying and linking the rearranged immunoglobulin heavy and light chain V-genes within single cells. Nucleic Acids Res. 20, 3831–3837.
Patterson, B., Till, M., Otto, P., Goolsby, C., Furtado, M., McBride, L., and Wolinsky, S. (1993) Detection of HIV-1 DNA and messenger RNA in individual cells by PCR-driven in situ hybridization and flow cytometry. Science 260, 976–979.
Gosden, J. and Lawson, D. (1995) In-situ cyclic amplification of oligonucleotide primed synthesis (cycling PRINS), in PCR Application Manual (Boehringer Mannheim Corp., ed.), Boehringer Mannheim Corp., Mannheim, Germany, pp. 115–118.
Komminoth, P., Adams, V., Long, A. A., Roth, J., Saremaslani, P., Flury, R., Schmid, M., and Heitz, P. U. (1994) Evaluation of methods for hepatitis C virus (HCV) detection in liver biopsies: comparison of histology, inmmunohistochemistry, in-situ hybridization, reverse transcriptase (RT) PCR and in-situ RT PCR. Pathol. Res. Pract. 190, 1017–1025.
Chen, R. H. and Fuggle, S. V. (1993) In situ cDNA polymerase chain reaction. A novel technique for detecting mRNA expression. Am. J. Pathol. 143, 1527–1534.
Komminoth, P. and Long, A. A. (1993) In-situ polymerase chain reaction. An overview of methods, applications and limitations of a new molecular technique. Virchows Arch. B. 64, 67–73.
Long, A. A., Komminoth, P., and Wolfe, H. J. (1992) Detection of HIV provirus by in situ polymerase chain reaction (letter). N. Engl. J Med. 327, 1529.
Long, A. A., Komminoth, P., Lee, E., and Wolfe, H. J. (1993) Comparison of indirect and direct in-situ polymerase chain reaction in cell preparations and tissue sections. Detection of viral DNA, gene rearrangements and chromosomal translocations. Histochemistry 99, 151–162.
Long, A. A. and Komminoth, P. (1995) In situ polymerase chain reaction: general methodology and recent advances, in In situ Polymerase Chain Reaction and Related Technology (Gu, J., ed.), Eaton Publishing, Natick, MA, pp. 23–34.
Komminoth, P. and Long, A. A. (1995) In-situ polymerase chain reaction-methodology, applications and non-specific pathways, in PCR Application Manual (Boehringer Mannheim Corp., ed.), Boehringer Mannheim Corp., Mannheim, Germany, pp. 97–106.
Komminoth, P., Heitz, P. U., and Long, A. A. (1994) In situ polymerase chain reaction: general methodology and recent advances. Verh. Dtsch. Ges. Pathol. 78, 146–152.
Chiu, K.-P., Cohen, S., Morris, D., and Jordan, G. (1992) Intracellular amplification of proviral DNA in tissue sections using the polymerase chain reaction. J. Histochem. Cytochem. 40, 333–341.
Komminoth, P., Long, A. A., Ray, R., and Wolfe, H. J. (1992) In situ polymerase chain reaction detection of viral DNA, single copy genes and gene rearrangements in cell suspensions and cytospins. Diagn. Mol. Pathol. 1, 85–97.
Embretson, J., Zupancic, M., Beneke, J., Till, M., Wolinsky, S., Ribas, J., Burke, A., and Haase, A. (1993) Analysis of human immunodeficiency virus-infected tissues by amplification and in situ hybridization reveals latent and permissive infections at single-cell resolution. Proc. Natl. Acad. Sci. USA 90, 357–361.
O’Leary, J. J., Browne, G., Landers, R. J., Crowley, M., Healy, I. B., Street, J. T., Pollock, A. M., Murphy, J., Johnson, M. I., Lewis, F. A., et al. (1994) The importance of fixation procedures on DNA template and its suitability for solution-phase polymerase chain reaction and PCR in situ hybridization. Histochem. J. 26, 337–346.
Bagasra, O., Sheshamma, T., Hansen, J., Bobiski, L., Saikumari, P., and Pomerantz, R. J. (1994) Application of in situ PCR methods in molecular biology. Special applications in electron microscopy, cytogenetics, and immunohistochemistry. Cell Vision 2, 61–70.
Long, A. A. (1991) Detection of gene rearrangements by in-situ PCR. Sixth Annual Workshop on Recent Advances in Molecular Pathology. Tufts New England Medical Center, Boston, MA, April 3–6, 1991.
Nuovo, G., Gallery, F., MacConnell, P., Becker, J., and Bloch, W. (1991) An improved technique for the in situ detection of DNA after polymerase chain reaction amplification. Am. J. Pathol. 139, 1239–1244.
Nuovo, G. J., Gorgone, G. A., MacConnell, P., Margiotta, M., and Gorevic, P. D. (1992) In situ localization of PCR-amplified human and viral cDNAs. PCR Methods Appl. 2, 117–123.
Erlich, H. A., Gelfand, D., and Sninsky, J. J. (1991) Recent advances in the polymerase chain reaction. Science 252, 1643–1651.
Komminoth, P., Long, A., and Wolfe, H. (1992) Comparison of in-situ polymerase chain reaction (in-situ PCR), in-situ hybridization (ISH) and polymerase chain reaction (PCR) for the detection of viral infection in fixed tissue. Patologia 25(Suppl.), 253.
Sällström, J. F., Zehbe, I., Alemi, M., and Wilander, E. (1993) Pitfalls of in situ polymerase chain reaction (PCR) using direct incorporation of labelled nucleotides. Anticancer Res. 13, 1153.
Komminoth, P. and Long, A. A. (1994) Questioning in-situ PCR. Detection of epidermal growth factor receptor mRNA in tissue sections from biopsy specimens using in-situ polymerase chain reaction (letter). Am. J. Pathol. 145, 742.
Sällström, J. F., Alemi, M., Spets, H., and Zehbe, I. (1994) Nonspecific amplification in in-situ PCR by direct incorporation of reporter molecules. Cell Vision 1, 243–251.
Zehbe, I., Sällström, J. F., Hacker, G. W., Hauser-Kronberger, C., Rylander, E., and Wilander, E. (1994) Indirect and direct in situ PCR for the detection of human papillomavirus. An evaluation of two methods and a double staining technique. Cell Vision 1, 163–167.
Teo, I. A. and Shaunak, S. (1995) PCR in situ: aspects which reduce amplification and generate false-positive results. Histochem. J. 27, 660–669.
Gold, R., Schmied, M., Rothe, G., Zischler, H., Breitschopf, H., Wekerle, H., and Lassmann, H. (1993) Detection of DNA fragmentation in apoptosis: application of in situ nick translation to cell culture systems and tissue sections. J. Histochem. Cytochem. 41, 1023–1030.
Wijsman, J., Jonker, R., Keijzer, R., van de Velde, C., Cornelisse, C., and van Dierendonck, J. (1993) A new method to detect apoptosis in paraffin sections. in situ end-labeling of fragmented DNA. J, Histochem. Cytochem. 41, 7–12.
Martinez, A., Miller, M. J., Quinn, K., Unsworth, E. J., Ebina, M., and Cuttitta, F. (1995) Non-radioactive localization of nucleic acids by direct in situ PCR and in situ RT-PCR in paraffin-embedded sections. J. Histochem. Cytochem. 43, 739–747.
Koch, J., Hindkjaer, J., Mogensen, J., Kölvraa, S., and Bolund, L. (1991) An improved method for chromosome-specific labeling of alpha satellite DNA in situ by using denatured double-stranded DNA probes as primers in a primed in situ labeling (PRINS) procedure. Genet. Anal. Tech. Appl. 8, 171–178.
Morita, M., Hachisuka, H., and Sasai, Y. (1994) Detection of genomic DNA with a high sensitivity in tissue sections using a two step cycling in situ PCR procedure. Kurume Med. J. 41, 215–220.
Staskus, K., Couch, L., Bitterman, P., Retzel, E., Zupancic, M., List, J., and Haase, A. (1991) In situ amplification of visna virus DNA in tissue sections reveals a reservoir of latently infected cells. Microb. Pathogen. 11, 67–76.
Zehbe, I., Hacker, G., Rylander, E., Sallstrom, J., and Wilander, E. (1992) Detection of single HPV copies in SiHa cells by in situ polymerase chain reaction (in situ PCR combined with immunoperoxidase and immunogold-silver staining [IGSS] techniques). Anticancer Res. 12, 2165–2168.
Bernard, C., Mougin, C., Bettinger, D., Didier, J. M., and Lab, M. (1994) Detection of human papillomavirus by in situ polymerase chain reaction in paraffin-embedded cervical biopsies. Mol. Cell Probes. 8, 337–343.
Bagasra, O., Hauptman, S., Lischner, H., Sachs, M., and Pomerantz, R. (1992) Detection of human immunodeficiency virus type 1 provirus in mononuclear cells by in situ polymerase chain reaction. N. Engl. J. Med. 326, 1385–1391.
Embretson, J., Zupancic, M., Ribas, J., Burke, A., Racz, P., Tenner-Racz, K., and Haase, A. (1993) Massive covert infection of helper T lymphocytes and macrophages by HIV during the incubation period of AIDS. Nature 362, 359–362.
Nuovo, G., Margiotta, M., MacConnell, P., and Becker, J. (1992) Rapid in situ detection of PCR-amplified HIV-1 DNA. Diagn. Mol. Pathol. 1, 98–102.
Nuovo, G., Forde, A., MacConnell, P., and Fahrenwald, R. (1993) In situ detection of PCR-amplified HIV-1 nucleic acids and tumor necrosis factor cDNA in cervical tissues. Am. J. Pathol. 143,1–9.
Wesselingh, S. L., Glass, J., McArthur, J. C., Griffin, J. W., and Griffin, D. E. (1994) Cytokine dysregulation in HIV-associated neurological disease. Adv. Neuroimmunol. 4, 199–206.
Re, M. C., Furlini, G., Gibellini, D., Vignoli, M., Ramazzotti, E., Lolli, E., Ranieri, S., and La, P. M. (1994) Quantification of human immunodeficiency virus type 1-infected mononuclear cells in peripheral blood of seropositive subjects by newly developed flow cytometry analysis of the product of an in situ PCR assay. J. Clin. Microbial. 32, 2152–2157.
Komminoth, P., Adams, V., Long, A. A., Roth, J., Saremaslani, P., Flury, R., Schmid, M., and Heitz, P. U. (1994) Comparison of histology, immunohistochemistry, RT-PCR, in situ hybridization, and in situ RT-PCR for demonstration of hepatitis C virus in paraffin-embedded liver biopsies. Verh. Dtsch. Ges. Pathol. 78, 220–225.
Nuovo, G. J., Lidonnici, K., MacConnell, P., and Lane, B. (1993) Intracellular localization of polymerase chain reaction (PCR)-amplified hepatitis C cDNA. Am. J. Surg. Pathol. 17, 683–690.
Lau, G. K. K., Fang, J. W. S., Wu, P. C., Davis, G. L., and Lau, J. Y. N. (1994) Detection of hepatitis C virus genome in formalin-fixed paraffin-embedded liver tissue by in situ reverse transcription polymerase chain reaction. J. Med. Virol. 44, 406–409.
Gressens, P. and Martin, J. R. (1994) HSV-2_DNA persistence in astrocytes of the trigeminal root entry zone: double labeling by in situ PCR and immunohistochemistry. J. Neuropathol. Exp. Neurol. 53, 127–135.
Mehta, A., Maggioncalda, J., Bagasra, O., Thikkavarapu, S., Saikumari, P., Valyi, N. T., Fraser, N. W., and Block, T. M. (1995) In situ DNA PCR and RNA hybridization detection of herpes simplex virus sequences in trigeminal ganglia of latently infected mice. Virology 206, 633–640.
Ramakrishnan, R., Levine, M., and Fink, D. J. (1994) PCR-based analysis of herpes simplex virus type 1 latency in the rat trigeminal ganglion established with a ribonucleotide reductase-deficient mutant. J. Virol. 68, 7083–7091.
Gressens, P., Langston, C., and Martin, J. R. (1994) In situ PCR localization of herpes simplex virus DNA sequences in disseminated neonatal herpes encephalitis. J. Neuropathol. Exp. Neurol. 53, 469–482.
Mitchell, W. J., Gressens, P., Martin, J. R., and DeSanto, R. (1994) Herpes simplex virus type 1 DNA persistence, progressive disease and transgenic immediate early gene promoter activity in chronic corneal infections in mice. J. Gen. Virol. 75, 1201–1210.
Kelleher, M. B., Galutira, D., Duggan, T. D., and Nuovo, G. J. (1994) Progressive multifocal leukoencephalopathy in a patient with Alzheimer’s disease. Diagn. Mol. Pathol. 3, 105–113.
Ueki, K., Richardson, E. P. J., Henson, J. W., and Louis, D. N. (1994) In situ polymerase chain reaction demonstration of JC virus in progressive multifocal leukoencephalopathy, including an index case. Ann. Neurol. 36, 670–673.
Zaki, S. R., Heneine, W., Coffield, L. M., Greer, P. W., Sinha, S. D., and Folks, T. M. (1994) In-situ polymerase chain reaction: applications and current limitations. AIDS 8, 1186–1188.
Walter, M. J., Lehky, T. J., Fox, C. H., and Jacobson, S. (1994) In situ PCR for the detection of HTLV-I in HAM/TSP patients. Ann. NY Acad. Sci. 724, 404–413.
Mankowski, J. L., Spelman, J. P., Ressetar, H. G., Strandberg, J. D., Laterra, J., Carter, D. L., Clements, J. E., and Zink, M. C. (1994) Neurovirulent simian immunodeficiency virus replicates productively in endothelial cells of the central nervous system in vivo and in vitro. J Virol. 68, 8202–8208.
Tsongalis, G. J., McPhail, A. H., Lodge, R. R. D., Chapman, J. F., and Silverman, L. M. (1994) Localized in situ amplification (LISA): a novel approach to in situ PCR. Clin. Chem. 40, 381–384.
Hu, M., Wang, W. L., and Zhao, Y. L. (1994) In situ PCR-gene hybridization method for detecting mycobacterium tuberculosis in tissue specimens. Chung Hua Chieh Ho Ho Hu Hsi Tsa Chih 17, 78,79.
Makowski, G. S., Aslanzadeh, J., and Hopfer, S. M. (1995) In situ PCR amplification of Guthrie card DNA to detect cystic fibrosis mutations (letter). Clin. Chem. 41, 477–479.
Gosden, J. and Hanratty, D. (1993) PCR in situ: a rapid alternative to in situ hybridization for mapping short, low copy number sequences without isotopes. Biotechniques 15, 78–80.
Palluy, O., Bendani, M., Vallat, J. M., and Rigaud, M. (1994) 12-lipoxygenase mRNA expression by cultured neurons. CR Acad. Sci. III 317, 133–818.
Nuovo, G. J., MacConnell, P. B., Simsir, A., Valea, F., and French, D. L. (1995) Correlation of the in situ detection of polymerase chain reaction-amplified metalloproteinase complementary DNAs and their inhibitors with prognosis in cervical carcinoma. Cancer Res. 55, 267–275.
Staecker, H., Cammer, M., Rubinstein, R., and VanDeWater, T. (1994) A procedure for RT-PCR amplification of mRNAs on histological specimens. Biotechniques 16, 76–80.
Heniford, B. W., Shum, S. A., Leonberger, M., and Hendler, F. J. (1993) Variation in cellular EGF receptor mRNA expression demonstrated by in situ reverse transcriptase polymerase chain reaction. Nucleic Acids Res. 21, 3159–3166.
Patel, V., Shum-Siu, A., Heniford, B., Wieman, T., and Hendler, F. (1994) Detection of epidermal growth factor receptor mRNA in tissue sections from biopsy specimens using in situ polymerase chain reaction. Am. J. Pathol. 144, 7–14.
Innis, M. A., Gelfand, H. D., Sniisky, J. J., and White, T. J. (1990) PCR Protocols. A Guide to Methods and Applications. Academic, San Diego, CA.
Nuovo, G., Gallery, F., Horn, R., MacConnell, P., and Bloch, W. (1993) Importance of different variables for enhancing in situ detection of PCR-amplified DNA. PCR Methods Appl. 2, 305–312.
Anderson, V. M. (1995) In situ PCR: new frontier for histopathologists, in In Situ Polymerase Chain Reaction and Related Technology (Gu, J., ed.), Eaton Publishing, Natick, MA, pp. 131–137.
Fahy, E., Kwoh, D. Y., and Gingeras, T. R. (1991) Self-sustained sequence replication (3SR): an isothermal transcription based amplification system alternative to PCR. PCR Methods Appl. 1, 25–33.
Zehbe, I., Hacker, G. W., Sällström, J. F., Rylander, E., and Wilander, E. (1994) Self-sustained sequence replication-based amplification (3SR) for the in-situ detection of mRNA in cultured cells. Cell Vision 1, 20–24.
Höfler, H., Pütz, B., Mueller, J., Neubert, W., Sutter, G., and Gais, P. (1995) In situ amplification of measles virus RNA by the self-sustained sequence replication reaction. Lab. Invest. 73, 577–585.
Tecott, L. H., Barchas, J. D., and Eberwine, J. H. (1988) In situ transcription: Specific synthesis of complementary DNA in fixed tissue sections. Science 240, 1661–1664.
Hacker, G. W. and Danscher, G. (1994) Recent advances in immunogold-silver staining—autometallography. Cell Vision 1, 102–109.
Raap, A. K., Van de Corput, M. P. C., Vervenne, R. A. W., van Gijlswijk, R. P. M., Tanke, H. J., and Wiegant, J. (1995) Ultra-sensitive FISH using peroxidase-mediated deposition of biotin-or fluorochrome tyramides. Hum. Mol. Genet. 4, 529–534.
Kolata, G. (1993) Tests show infection by AIDS virus affects greater share of cells. The New York Times, January 5, C3.
Komminoth, P. and Long, A. A. (1995) In situ polymerase chain reaction and its applications to the study of endocrine diseases. Endocr. Pathol. 6, 167–171.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1997 Humana Press Inc, Totowa, NJ
About this protocol
Cite this protocol
Long, A.A., Komminoth, P. (1997). In Situ PCR. In: Gosden, J.R. (eds) PRINS and In Situ PCR Protocols. Methods in Molecular Biology™, vol 71. Humana Press. https://doi.org/10.1385/0-89603-395-3:141
Download citation
DOI: https://doi.org/10.1385/0-89603-395-3:141
Publisher Name: Humana Press
Print ISBN: 978-0-89603-395-5
Online ISBN: 978-1-59259-557-0
eBook Packages: Springer Protocols