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Signal Amplification for DNA and mRNA

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In Situ Hybridization Protocols

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 123))

Abstract

In situ hybridization (ISH) permits the localization of specific unique or repeated DNA and RNA sequences at the level of individual cells (1-4). It has significantly advanced the study of gene structure and expression, and, in addition to morphological identification of cell types involved, ISH also allows some quantification of observations, e.g., with respect to tumor burden or viral load. Despite its high degree of detection specificity, the technique still does not allow the routine detection of DNA sequences less than 5 kb in size, and in the case of tissue sections the detection sensitivity is even more limited. The threshold levels for mRNA detection are more difficult to determine, with the reported sensitivity limits of 1–20 copies of mRNA per cell being achieved only in the most sensitive protocols (5,6,6a).

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References

  1. Joos, S., Fink, T. M., Rätsch, A., and Lichter, P. (1994) Mapping and chromosome analysis: the potential of fluorescence in situ hybridization. J. Biotechnol. 35, 135–153.

    Article  PubMed  CAS  Google Scholar 

  2. Dirks, R. W. (1996) RNA molecules lighting up under the microscope. Histochem. Cell Biol. 106, 151–166.

    Article  PubMed  CAS  Google Scholar 

  3. Hopman, A. H. N., Voorter, C. E. M., Speel, E. J. M., and Ramaekers, F. C. S. (1997) In situ hybridization and comparative genomic hybridization, in Cytogenetic Cancer Markers (Wolman, S. R. and Sell, S., eds.), Humana Press, Totowa, NJ, pp. 45–69.

    Google Scholar 

  4. McNicol, A. M. and Farquharson, M. A. (1997) In situ hybridization and its diagnostic applications in pathology. J. Pathol. 182, 250–261.

    Article  PubMed  CAS  Google Scholar 

  5. Höfler, H., Childers, H., Montminy, M. R., Lechan, R. M., Goodmann, R. H., and Wolfe, H. J. (1986) In situ hybridization methods for the detection of somatostatin mRNA in tissue sections using antisense RNA probes. Histochem. J. 18, 597–604.

    Article  Google Scholar 

  6. Komminoth, P. (1996) Detection of mRNA in tissue sections using digoxigenin-labeled RNA and oligonucleotide probes, in Nonradioactive In Situ Hybridization Application Manual. Boehringer, Mannheim, Germany, pp. 126–135.

    Google Scholar 

  7. Femino, A. M., Fay, F. S., Fogarty, K., and Singer, R. H. (1998) Visualization of single RNA transcripts in situ. Science 280, 585–590.

    Article  PubMed  CAS  Google Scholar 

  8. Lloyd, R. V. and Jin, L. (1995) In situ hybridization analysis of chromogranin A and B mRNAs in neuroendocrine tumors with digoxigenin-labeled oligonucle-otide probe cocktails. Diagn. Mol. Pathol. 4, 143–151.

    Article  PubMed  CAS  Google Scholar 

  9. Trembleau, A. and Bloom, F. E. (1995) Enhanced sensitivity for light and electron microscopic in situ hybridization with multiple simultaneous non-radioactive oligodeoxynucleotide probes. J. Histochem. Cytochem. 43, 829–841.

    PubMed  CAS  Google Scholar 

  10. 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.

    Article  PubMed  CAS  Google Scholar 

  11. Nuovo, G. (1992) PCR In Situ Hybridization. Raven Press, New York.

    Google Scholar 

  12. 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 Cell Pathol. 64, 67–73.

    Article  CAS  Google Scholar 

  13. 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.

    CAS  Google Scholar 

  14. 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.

    PubMed  Google Scholar 

  15. 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.

    PubMed  CAS  Google Scholar 

  16. Terkelsen, C., Koch, J., Kolvraa, S., Hindkjaer, J., Pedersen, S., and Bolund, L. (1993) Repeated primed in situ labeling: formation and labeling of specific DNA sequences in chromosomes and nuclei. Cytogenet. Cell Genet. 63, 235–237.

    Article  PubMed  CAS  Google Scholar 

  17. Troyer, D. L., Goad, D. W., Xie, H., Rohrer, G. A., Alexander, L. J., and Beattle, C. W. (1994) Use of direct in situ single-copy (DISC) PCR to physically map five porcine microsatellites. Cytogenet. Cell Genet. 67, 199–204.

    Article  PubMed  CAS  Google Scholar 

  18. Kerstens, H. M., Poddighe, P. J., and Hanselaar, A. G. (1995) A novel in situ hybridization signal amplification method based on the deposition of biotinylated tyramine. J. Histochem. Cytochem. 43, 347–352.

    PubMed  CAS  Google Scholar 

  19. 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.

    Article  PubMed  CAS  Google Scholar 

  20. Komminoth, P. and Werner, M. (1997) Target and signal amplification: approaches to increase the sensitivity of in situ hybridization. Histochem. Cell Biol. 108, 325–333.

    Article  PubMed  CAS  Google Scholar 

  21. Speel, E. J. M., Saremaslani, P., Roth, J., Hopman, A. H. N., and Komminoth, P. (1998) Improved mRNA in situ hybridization on formaldehyde-fixed and paraffin-embedded tissue using signal amplification with different haptenized tyramides. Histochem. Cell Biol. 110, 571–577.

    Article  PubMed  CAS  Google Scholar 

  22. Speel, E. J. M., Hopman, A. H. N., and Komminoth, P. (1999) Amplification methods to increase the sensitivity of in situ hybridization: Play CARD(S). J. Histochem. Cytochem. 47, 281–288.

    PubMed  CAS  Google Scholar 

  23. Speel, E. J. M., Ramaekers, F. C. S., and Hopman, A. H. N. (1997) Sensitive multicolor fluorescence in situ hybridization using catalyzed reporter deposition (CARD) amplification. J. Histochem. Cytochem. 45, 1439–1446.

    PubMed  CAS  Google Scholar 

  24. Gosden, J. and Lawson, D. (1995) In-situ cyclic amplification of oligonucleotide primed synthesis (cycling PRINS), in PCR Application Manual. Boehringer, Mannheim, Germany, pp. 115–118.

    Google Scholar 

  25. 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.

    PubMed  CAS  Google Scholar 

  26. 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, immunohistochemistry, in-situ hybridization, reverse transcriptase (RT) PCR and in-situ RT PCR. Pathol. Res. Pract. 190, 1017–1025.

    PubMed  CAS  Google Scholar 

  27. 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.

    Article  CAS  Google Scholar 

  28. 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.

    Google Scholar 

  29. Komminoth, P. and Long, A. A. (1995) In-situ polymerase chain reaction—methodology, applications and non-specific pathways, in PCR Application Manual. Boehringer, Mannheim, Germany, pp. 97–106.

    Google Scholar 

  30. Höfler, H. (1993) In situ polymerase chain reaction: toy or tool? Histochemistry 99, 103–104.

    Article  PubMed  Google Scholar 

  31. 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.

    Article  PubMed  CAS  Google Scholar 

  32. Sällström, J. F., Zehbe, I., Alemi, M., and Wilander, E. (1993) Pitfalls of in situ polymerase chain reaction (PCR) using direct incorporation of labeled nucleotides. Anticancer Res. 13, 1153.

    PubMed  Google Scholar 

  33. Long, A. A. and Komminoth, P. (1996) In situ polymerase chain reaction: an overview, in Methods in Molecular Biology, vol. XX, Protocols for PRINS and In Situ PCR (Gosden, J. R., ed.), Humana Press, Totowa, NJ, pp. 141–161.

    Google Scholar 

  34. Lizardi, P. M., Huang, X., Zhu, Z., Bray-Ward, P., Thomas, D. C., and Ward, D. C. (1998) Mutation detection and single-molecule counting using insothermal rolling-circle amplification. Nat. Genet. 199, 225–232.

    Article  Google Scholar 

  35. Bobrow, M. N., Harris, T. D., Shaughnessy, K. J., and Litt, G. J. (1989) Catalyzed reporter deposition, a novel method of signal amplification. Application to immunoassays. J. Immunol. Methods 125, 279–285.

    Article  PubMed  CAS  Google Scholar 

  36. Adams, J. C. (1992) Biotin amplification of biotin and horseradish peroxidase signals in histochemical stains. J. Histochem. Cytochem. 40, 1457–1463.

    PubMed  CAS  Google Scholar 

  37. Berghorn, K. A., Bonnett, J. H., and Hoffman, G. E. (1994) cFos immunoreactivity is enhanced with biotin amplification. J. Histochem. Cytochem. 42, 1635–1642.

    PubMed  CAS  Google Scholar 

  38. Merz, H., Malisius, R., Mannweiler, S., Zhou, R., Hartmann, W., Orscheschek, K., Moubayed, P., and Feller, A. C. (1995) ImmunoMax. A maximized immunohistochemical method for the retrieval and enhancement of hidden antigens. Lab. Invest. 73, 149–156.

    PubMed  CAS  Google Scholar 

  39. Werner, M., Von Wasielewski, R., and Komminoth, P. (1996) Antigen retrieval, signal amplification and intensification in immunohistochemistry. Histochem. Cell Biol. 105, 253–260.

    Article  PubMed  CAS  Google Scholar 

  40. Von Wasielewski, R., Mengel, M., Gignac, S., Wilkens, L., Werner, M., and Georgii, A. (1997) Tyramine amplification technique in routine immunohistochemistry. J. Histochem. Cytochem. 45, 1455–1459.

    Google Scholar 

  41. Poddighe, P. J., Bulten, J., Kerstens, H. M. J., Robben, J. C. M., Melchers, W. J. G., and Hanselaar, A. G. J. M. (1996) Human papilloma virus detection by in situ hybridisation signal amplification based on biotinylated tyramine deposition. Clin. Mol. Pathol. 49, M340–344.

    Article  PubMed  CAS  Google Scholar 

  42. Macechko, P. T., Krueger, L., Hirsch, B., and Erlandsen, S. L. (1997) Comparison of immunologic amplification vs enzymatic deposition of fluorochrome-conjugated tyramide as detection systems for FISH. J. Histochem. Cytochem. 45, 359–363.

    PubMed  CAS  Google Scholar 

  43. Schmidt, B. F., Chao, J., Zhu, Z., Debiasio, R. L., and Fisher, G. (1997) Signal amplification in the detection of single-copy DNA and RNA by enzyme-catalyzed deposition (CARD) of the novel fluorescent reporter substrate Cy3. 29-tyramide. J. Histochem. Cytochem. 45, 365–373.

    PubMed  CAS  Google Scholar 

  44. Van Gijlswijk, R. P. M., Zijlmans, H. J. M. A. A., Wiegant, J., Bobrow, M. N., Erickson, T. J., Adler, K. E., Tanke, H. J., and Raap, A. K. (1997) Fluorochrome-labeled tyramides: use in immunocytochemistry and fluorescence in situ hybridization. J. Histochem. Cytochem. 45, 375–382.

    PubMed  Google Scholar 

  45. Zehbe, I., Hacker, G. W., Su, H., Hauser-Kronberger, C., Hainfeld, J. F., and Tubbs, R. (1997) Sensitive in situ hybridization with catalyzed reporter deposition, streptavidin-nanogold, and silver acetate autometallography. Am. J. Pathol. 150, 1553–1561.

    PubMed  CAS  Google Scholar 

  46. Hopman, A. H. N., Ramaekers, F. C. S., and Speel, E. J. M. (1998) Rapid synthesis of biotin-, digoxigenin-, trinitrophenyl-, and fluorochrome-labeled tyramides and their application for in situ hybridization using CARD-amplification. J. Histochem. Cytochem. 46, 771–777.

    PubMed  CAS  Google Scholar 

  47. Van Gijlswijk, R. P. M., Wiegant, J., Vervenne, R., Lasan, R., Tanke, H. J., and Raap, A. K. (1996) Horseradish peroxidase-labeled oligonucleotides and fluorescent tyramides for rapid detection of chromosome-specific repeat sequences. Cytogenet. Cell Genet. 75, 258–262.

    Article  PubMed  Google Scholar 

  48. Lichter, P. and Ried, T. (1994) Molecular analysis of chromosome aberrations: in situ hybridization, in Methods in Molecular Biology, vol. 29. Chromosome Analysis Protocols (Gosden, J. R., ed.), Humana Press, Totowa, NJ, pp. 449–478.

    Google Scholar 

  49. Dutrillaux, B. and Viegas-Pequignot, E. (1985) High resolution R-and G-banding in the same preparation. Hum. Genet. 57, 93–95.

    Google Scholar 

  50. Speel, E. J. M., Schutte, B., Wiegant, J., Ramaekers, F. C. S., and Hopman, A. H. N. (1992) A novel fluorescence detection method for in situ hybridization, based on the alkaline phosphatase-fast red reaction. J. Histochem. Cytochem. 40, 1299–1308.

    PubMed  CAS  Google Scholar 

  51. Spowart, G. (1994) Mitotic metaphase chromosome preparation from peripheral blood for high resolution, in Methods in Molecular Biology, vol. 29. Chromosome Analysis Protocols (Gosden, J. R., ed.), Humana Press, Totowa, NJ, pp. 1–10.

    Google Scholar 

  52. Ross, F. (1994) Chromosome preparation from hematological malignancies, in Methods in Molecular Biology, vol. 29. Chromosome Analysis Protocols (Gosden, J. R., ed.), Humana Press, Totowa, NJ, pp. 11–25.

    Google Scholar 

  53. Langer, P. R., Waldrop, A. A., and Ward, D. C. (1981) Enzymatic synthesis of biotin-labeled polynucleotides: novel nucleic acid affinity probes. Proc. Natl. Acad. Sci. USA 78, 6633–6637.

    Article  PubMed  CAS  Google Scholar 

  54. Boehringer (1996) Nonradioactive In Situ Hybridization Application Manual. 2nd ed. Boehringer, Mannheim, Germany.

    Google Scholar 

  55. Speel, E. J. M., Kamps, M., Bonnet, J., Ramaekers, F. C. S., and Hopman, A. H. N. (1993) Multicolour preparations for in situ hybridization using precipitating enzyme cytochemistry in combination with reflection contrast microscopy. Histochemistry 100, 357–366.

    Article  PubMed  CAS  Google Scholar 

  56. Speel, E. J. M., Jansen, M. P. H. M., Ramaekers, F. C. S., and Hopman, A. H. N. (1994) A novel triple-color detection procedure for brightfield microscopy, combining in situ hybridization with immunocytochemistry. J. Histochem. Cytochem. 42, 1299–1307.

    PubMed  CAS  Google Scholar 

  57. Speel, E. J. M., Ramaekers, F. C. S., and Hopman, A. H. N. (1995) Detection systems for in situ hybridization, and the combination with immunocytochemistry. Who is still afraid of red, green and blue? Histochem. J. 27, 833–858.

    PubMed  CAS  Google Scholar 

  58. Hopman, A. H. N., Speel, E. J. M., Voorter, C. E. M., and Ramaekers, F. C. S. (1995) Probe labelling methods, in Non-Isotopic Methods in Molecular Biology: A Practical Approach (Levy, E. R. and Herrington, C. S., eds.), IRL Press, Oxford University Press, Oxford, UK, pp. 1–24.

    Google Scholar 

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Author information

Authors and Affiliations

  1. Department of Pathology, University Hospital Zürich, Zürich, Switzerland

    Ernst J. M. Speel & Paul Komminoth

  2. Department of Molecular Cell Biology and Genetics, University Maastricht, Maastricht, The Netherlands

    Anton H. N. Hopman

Authors
  1. Ernst J. M. Speel
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  2. Anton H. N. Hopman
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  3. Paul Komminoth
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Editors and Affiliations

  1. RMIT University Bundoora, Victoria, Australia

    Ian A. Darby

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© 2000 Humana Press Inc.

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Speel, E.J.M., Hopman, A.H.N., Komminoth, P. (2000). Signal Amplification for DNA and mRNA. In: Darby, I.A. (eds) In Situ Hybridization Protocols. Methods in Molecular Biology™, vol 123. Humana Press. https://doi.org/10.1385/1-59259-677-0:195

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  • DOI: https://doi.org/10.1385/1-59259-677-0:195

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-686-4

  • Online ISBN: 978-1-59259-677-5

  • eBook Packages: Springer Protocols

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