Advertisement

PCR Protocols pp 433-444 | Cite as

Direct and Indirect In Situ PCR

  • Klaus Hermann Wiedorn
  • Torsten Goldmann
Part of the Methods in Molecular Biology™ book series (MIMB, volume 226)

Abstract

In recent years, the development of in situ technologies has made good progress. In situ hybridization (ISH) has become an important tool and has enabled the pathologist to demonstrate infectious pathogens or mRNAs in tissue sections or cytospins without destruction of morphology, thus enabling the assignment of signals to individual cells or cell compartments (1, 2, 3, 4, 5, 6, 7, 8, 9).

Keywords

Dextran Sulfate Nitroblue Tetrazolium Chloride Detection Buffer Buffer Paraformaldehyde Endogenous Priming 
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.
    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.PubMedGoogle Scholar
  2. 2.
    Goldmann, T., Becher, B., Wiedorn, K. H., Pirow, R., Deutschbein, M. E., Vollmer, E., et al. (1999) Epipodite and fat cells as sites of hemoglobin synthesis in the branchiopod crustacean Daphnia magna. Histochem. Cell Biol. 112, 335–339.PubMedGoogle Scholar
  3. 3.
    Heniford, B. W., Shum-Siu, A., Leonberger, M., and Hendler, F. J. (1993) Variation in cellular, E. G.F receptor mRNA expression demonstrated by in situ reverse transcriptase polymerase chain reaction. Nucleic Acids Res. 21, 3159–3166.PubMedCrossRefGoogle Scholar
  4. 4.
    Long, A. A., Komminoth, P., and Wolfe, H. J. (1992) Detection of HI.V provirus by in situ polymerase chain reaction. N. Engl. J. Med. 327, 1529.PubMedCrossRefGoogle Scholar
  5. 5.
    Nuovo, G. J. (1994) PCR in Situ Hybridization Protocols and Applications, Second Ed., Raven Press, New York.Google Scholar
  6. 6.
    Patel, V. G., Shum-Siu, A., Heniford, B. W., Wieman, T. J., and Hendler, F. J. (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.PubMedGoogle Scholar
  7. 7.
    Snijders, P. J. F., van den Brule, A. J. C., Schrijnemakers, H. F. J., Snow, G., Meijer, C. J. L. M., and Walboomers, J. M. M. (1990) The use of general primers in the polymerase chain reaction permits detection of a broad spectrum of human papillomavirus genotypes. J. Gen. Virol. 71, 173–181.PubMedCrossRefGoogle Scholar
  8. 8.
    Wiedorn, K. H., Kühl, H., Galle, J., Caselitz, J., and Vollmer, E. (1999) Comparison of insitu hybridization, direct and indirect in-situ PCR as well as tyramide signal amplification for the detection of HPV. Histochem. Cell Biol. 111, 89–95.Google Scholar
  9. 9.
    Wiedorn, K. H., Goldmann, T., Kühl, H., Galle, J., and Vollmer, E. (1999) GenPoint™: A new biotinyl tyramide based signal amplification system for significantly increasing sensitivity of in situ hybridization (ISH). Elec. J. Pathol. Histol. 994–999.Google Scholar
  10. 9a.
    Furuta, Y., Shinohara, T., Sano, K., Meguro, M., and Nagashima, K. (1990) In situ hybridization with digoxigenin-labelled DNA probes for detection of viral genomes. J. Clin. Pathol. 43, 806–809.PubMedCrossRefGoogle Scholar
  11. 10.
    Allan, G. M., Todd, D., Smyth, J. A., Mackie, D. P., Burns, J., and McNulty, M. S. (1989) In situ hybridization: an optimised detection protocol for a biotinylated DNA probe renders it more sensitive than a comparable 35S-labeled probe. J. Virol. Methods 24, 181–190.PubMedCrossRefGoogle Scholar
  12. 11.
    Burns, J., Graham, A., Frak, C., Fleming, F., Evans, M., and McGee, J. (1987) Detection of low copy human papilloma virus DNA and mRNA in routine paraffin sections of cervix by non-isotopic in situ hybridization. J. Clin. Pathol. 40, 858–864.PubMedCrossRefGoogle Scholar
  13. 12.
    Furuta, Y., Shinohara, T., Sano, K., Meguro, M., and Nagashima, K. (1990) In situ hybridization with digoxigenin-labelled DNA probes for detection of viral genomes. J. Clin. Pathol. 43, 806–809.PubMedCrossRefGoogle Scholar
  14. 13.
    Nuovo, G. J. and Richart, R. M. (1989) A comparison of biotin and 35S-based in situ hybridization methodologies for detection of human papilloma-virus DNA. Lab. Invest. 61, 471–476.PubMedGoogle Scholar
  15. 14.
    Syrjänen, S., Partanen, P., Mäntyjärvi, R., and Syrjanen K. (1988) Sensitivity of in situ hybridization techniques using biotin-and 35S-labeled human papillomavirus (HPV) DNA probes. J. Virol. Methods 19, 225–238.PubMedCrossRefGoogle Scholar
  16. 15.
    Wiedorn, K. H., Lang, D., Köhl, H., Galle, J., and Vollmer, E. (1999) Sensitive in situ detection of modulated expression levels of interleukin 6 (IL6) and interleukin 8 (IL8) in bronchial epithelium and leucocytes using cDNA specific primers and direct RT in-Situ-PCR. Elec. J. Pathol. Histol. 993–996.Google Scholar
  17. 16.
    Herrington, C. S., Burns, J., Graham, A. K., Evans, M., and McGee, J. O. (1989) Interphase cytogenetics using biotin and digoxigenin labeled probes I: relative sensitivity of both reporter molecules for detection of HPV 16 in CaSki cells. J. Clin. Pathol. 42, 592–600.PubMedCrossRefGoogle Scholar
  18. 17.
    Holm, R., Karlsen, F., and Nesland, J. M. (1992) In situ hybridization with nonisotopic probes using different detection systems. Mod. Pathol. 5, 315–319.PubMedGoogle Scholar
  19. 18.
    Lassus, J., Niemi, K.-M., Marjamaki, A., Syrjanen, S., Kartamaa, M., Lehmus, A., et al. (1992) Comparison of four in situ hybridization methods, based on digoxigenin-and biotin-labelled probes, in detecting HPV DNA in male condylomata acuminata. Int. J. STD AIDS 3, 196–203.PubMedGoogle Scholar
  20. 19.
    Mifflin, T. E., Bowdwn, J., Lovell, M. A., Burns, D. E., Hayden, F. G., Graschel, D. H. M, et al. (1987) Comparison of radioactive and biotinylated DNA probes for the detection of cytomegalovirus. Clin. Biochem. 20, 231–235.PubMedCrossRefGoogle Scholar
  21. 20.
    Niedobitek, G., Finn, T., Herbst, H., and Stein, H. (1989) In situ hybridization using biotinylated probes. An evaluation of different detection systems. Pathol. Res. Pract. 184, 343–348.PubMedGoogle Scholar
  22. 21.
    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 Archiv. 64, 67–73.PubMedCrossRefGoogle Scholar
  23. 22.
    Komminoth, P. and Long, A. A. (1995) In situ polymerase chain reaction—methodology, applications and nonspecific pathways, in PCR Applications Manual, Boehringer Mannheim, Mannheim, pp. 97–106.Google Scholar
  24. 23.
    Long, A. A., Komminoth, P., 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, 51–162.CrossRefGoogle Scholar
  25. 24.
    Söllström, 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, 1153–1154.Google Scholar
  26. 25.
    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.PubMedCrossRefGoogle Scholar
  27. 26.
    Komminoth, P., Adams, V., Long, A. A., Roth, J., Saremaslani, P, Flury, R., et al. (1994) Evaluation of methods for hepatitis C virus (HCV) detection in liver biopsies: Comparison of histology, immunohistochemistry, in situ hybridization, reverse (RT) PCR and in situ RT PCR. Pathol. Res. Pract. 190, 1017–1025.PubMedGoogle Scholar
  28. 27.
    Wiedorn, K. H., Schaaf, B., Kühl, H., Spuck, S., Galle, J., Dalhoff, K., et al. (1999) Granulocyte Colony Stimulating Factor (G-CSF) induced modulation of cytokine expression: A semiquantitative in situ analysis using target amplification by reverse transcriptase in situ PCR (RT IS PCR) as well as signal amplification by in situ hybridization (ISH) with catalyzed reporter deposition (CARD). Verhandlungen der Deutschen Gesellschaft für Zytologie 21, 63–65.Google Scholar
  29. 28.
    Wiedorn, K. H., Goldmann, T., Kühl, H., Rohrer, Ch., and Vollmer, E. (2000) Single-copy virus detection by GenPoint in situ hybridisation on the Eppendorf Mastercycler. BioTech Int. 12, 12–13.Google Scholar
  30. 29.
    Wiedorn, K. H. (1996) In situ Hybridisierungshandbuch zum MISHA in situ Thermocycler, in Misha Multipler In-Situ-Hybridisierungsautomat für die In-Situ-Hybridisierung und Polymerase-Kettenreaktion (PCR), Shandon, Frankfurt.Google Scholar
  31. 30.
    Stapleton, M. J., Levin, M. C., and Jacobson, S. (1994) DNA Amplification within cells on slides: Advances in evaporation and temperature control. Cell Vision 1, 177–181.Google Scholar

Copyright information

© Humana Press Inc. 2003

Authors and Affiliations

  • Klaus Hermann Wiedorn
    • 1
  • Torsten Goldmann
    • 2
  1. 1.Institut für PathologieKatharinenhospital StuttgartStuttgartGermany
  2. 2.Clinical and Experimental PathologyResearch Center BorstelBorstelGermany

Personalised recommendations