Skip to main content
SpringerLink
Log in

In Situ RT-PCR and Hybridization Techniques

  • Protocol
Immunocytochemical Methods and Protocols

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 115))

Abstract

In situ polymerase chain reaction (PCR) is a very powerful tool, which enhances our ability to detect minute quantities of a rare, single copy number, target nucleic acid sequences in freshly frozen or paraffin-embedded intact cells or tissue sections (1–10). In 1986, the introduction of PCR methods opened new horizons and revolutionized research in all areas of molecular biology (11,12). Dr. Hasse and his coworkers in 1990 used multiple primers and successfully amplified the target nucleic acid sequences in intact cells by combining a traditional in situ hybridization protocol with a powerful PCR technology (13).

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Nuovo, G. J. (1997) PCR in situ hybridization: protocols and applications, 3rd ed. Lippincott-Raven, Philadelphia, PA.

    Google Scholar 

  2. 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. Diag. Mol. Pathol. 1, 85–97.

    CAS  Google Scholar 

  3. 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. Mod. Pathol. 4, 124A.

    Google Scholar 

  4. Nuovo, G. J., 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.

    PubMed  CAS  Google Scholar 

  5. 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 

  6. 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.

    PubMed  CAS  Google Scholar 

  7. Chieu, K. P., Cohen, S. H., Morris, D. W., and Jordan, G. W. (1992) Intracellular amplification of proviral DNA in tissue sections using the polymerase chain reaction. J. Histochem. Cytochem. 40, 333–341.

    Google Scholar 

  8. Spann, W., Pachmann, K., Zabnienska, H., Pielmeier, A., and Emmerich, B. (1991) In-situ amplification of single copy gene segments in individual cells by polymerase chain reaction. Infection 19, 242–244.

    Article  PubMed  CAS  Google Scholar 

  9. Bagasra, O., Sechamma, T., and Promerantz, R. J. (1993) Polymerase chain reaction in-situ: intracellular amplification and detection of HIV-1 proviral DNA and other specific genes. J. Immunol. Meth. 158, 131–145.

    Article  CAS  Google Scholar 

  10. Komminoth, P. and Long, A. A. (1993) In situ polymerase chain reaction. An overview of methods, applications and limitation of a new molecular technique. Virchows Arch. 64, 67–73.

    Article  CAS  Google Scholar 

  11. Rodu, B. (1990) The polymerase chain reaction: the revolution within. Am. J. Med. Sci. 299, 210–216.

    Article  PubMed  CAS  Google Scholar 

  12. Eeles, R. A., Warren, W., and Stamps, A. (1992) The PCR revolution. Eur. J. Cancer 28, 289–293.

    Article  PubMed  CAS  Google Scholar 

  13. 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 

  14. Zevallos, E., Anderson, V., Novakoski, M., Bard, E., and Gu, J. (1994) Detection of human retinoblastoma gene expression by rTth-driven reverse transcribed in situ PCR. Cell Vision 1, 88.

    Google Scholar 

  15. Kellogg, D. E., Rybalkin, Y., and Chen, S. (1994) Taq-start antibody: hot start PCR facilitated by a neutralizing monoclonal antibodies directed against Taq DNA polymerase Biotechniques 16, 1134–1137.

    PubMed  CAS  Google Scholar 

  16. Simsor, S. and Nuovo, G. J. (1995) PCR and RT-PCR in situ hybridization: application in viral detection. Trends Biotechnol. 11, 13–23.

    Google Scholar 

  17. Embertson, J., Zupanic, M., Beneke, T., Till, M., Wolinsky, S., Ribas, J. L., Burke, A., and Haase, A. T. (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.

    Article  Google Scholar 

  18. Nuovo, G. J., Becker, J., Simsir, A., Morgiotta, M., Khalife, G., and Shevchuk, M. (1994) HIV-1 nucleic acids localize to the spermatogonia and their progeny: a study by polymerase chain reaction in situ hybridization. Am. J. Pathol. 144, 1142–1148.

    PubMed  CAS  Google Scholar 

  19. Kato, J., Hirata, S., Hagihara, K., Osada, T., Hirai, M., and Ikagami, J. (1992) Gene expression of steroid hormone receptors in brain and peripheral tissues relative to reproduction. Acta Histochem. Cytochem. 25, 667–680.

    CAS  Google Scholar 

  20. Lev-Lehman, E., Ginzberg, D., Hornreich, G., Ehrlich, G., Meshorer, A., Eckstein, F., Soreq, H., and Zakut, H. (1994) Antisense inhibition of acetylcholinesterase gene expression causes transient hematopoietic alterations in vivo. Gene Therapy 1, 127–135.

    PubMed  CAS  Google Scholar 

  21. Shroyer, K. R., Brooks, C. G., Markham, N. E., and Shroyer, A. L. (1995) Detection of human papillomavirus in anorectal squamous cell carcinoma. Am. J. Clin. Pathol. 104, 299–305.

    PubMed  CAS  Google Scholar 

  22. Kumazaki, T., Hamada, K., and Mitsui, Y. (1994) Detection of mRNA expression in a single cell by direct RT-PCR. Biotechniques 16, 1017–1019.

    PubMed  CAS  Google Scholar 

  23. Chiocchia, G. and Smith, K. A. (1997) Highly sensitive method to detect mRNAs in individual cells by direct RT-PCR using Tth DNA polymerase. Biotechniques 22, 312–314.

    PubMed  CAS  Google Scholar 

  24. Greenwood, D., Yao, Wp, and Housley, G. D. (1997) Expression of the P2X2 receptor subunit of the ATP-gated ion channel in the retina. Neuroreport. 8, 1083–1088.

    Article  PubMed  CAS  Google Scholar 

  25. Sanno, N., Jin, L., Qian, X., Osamura, R. Y., Scheithauer, B. W., Kovacs, K., and Lloyd, R. V. (1997) Gonadotropin-releasing hormone and gonadotropin-releasing hormone receptor messenger ribonucleic acids expression in nontumorous and neoplastic pituitaries. J. Clin. Endocrinol. Metab. 82, 1974–1982.

    Article  PubMed  CAS  Google Scholar 

  26. Amarsham International Plc. (1994) A guide to radioactive & non-radioactive in situ hybridization systems.

    Google Scholar 

  27. Giang, Gu. (1995) In situ PCR and related technology. Eaton Publishing Co.

    Google Scholar 

  28. Isaacson, S. H., Asher, D. M., Gibbs, C. J., and Gajdusek, D. C. (1994) In situ RT-PCR amplification in archival brain tissue. Cell Vision 1, 25–28.

    CAS  Google Scholar 

  29. Sebbelov, A. M., Svendsen, C., Jensen, H., Kjaer, S. K., and Norrild, B. (1994) Prevalence of HPV in premalignant and malignant cervical lesions in Greenland and Denmark: PCR and in situ hybridization analysis on archival material. Res. Virol. 145, 83–92.

    Article  PubMed  CAS  Google Scholar 

  30. Drut, R. M., Day, S., Drut, R., and Meisner, L. (1994) Demonstration of Epstein-Barr viral DNA in paraffin-embedded tissues of Burkitt’s lymphoma from Argentina using the polymerase chain reaction and in situ hybridization. Ped. Pathol. 14, 101–109.

    Article  CAS  Google Scholar 

  31. 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.

    PubMed  CAS  Google Scholar 

  32. 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., Mohamdee, O., Cullinane, C., and Doyle, C. T. (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.

    Article  PubMed  Google Scholar 

  33. Greer, C. E., Lund, J. K., and Manos, M. M. (1991) PCR amplification from paraffin-embedded tissues: recommendation on fixatives for long term storage and prospective studies. PCR Meth. Appl. 95, 117–124.

    CAS  Google Scholar 

  34. Greer, C. E., Peterson, S. L., Kiviar, 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.

    PubMed  CAS  Google Scholar 

  35. Bagasra, O., Seshamma, T., Romerantz, R., and Hansen, J. (1995) In situ PCR* Hybridization to detect low-abundance nucleic acid targets. Current protocols in molecular biology, vol. 2, sections 14.8.1–14.8.23 (Suppl. 31).

    Google Scholar 

  36. 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, 1527–2534.

    PubMed  CAS  Google Scholar 

  37. Teo, I. A. and Shaunak, S. (1995) PCR in situ: aspects which reduce amplification and generate false-positive results. Histochem. J. 27, 660–669.

    PubMed  CAS  Google Scholar 

  38. Pallansch, L., Beswick, H., Talian, J., and Zelenka, P. (1990) Use of an RNA folding algorithm to choose regions for amplification by the polymerase chain reaction. Anal. Biochem. 185, 57–62.

    Article  PubMed  CAS  Google Scholar 

  39. Freeman, W. M., Vrana, S. L., and Vrana, K. E. (1996) Use of elevated reverse transcription reaction temperatures in RT-PCR. Biotechniques 20, 782–783.

    PubMed  CAS  Google Scholar 

  40. 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 

  41. Salstrom, J., Alemi, M., and Wilander, E. (1993) Pitfalls of in situ PCR using direct incorporation of labeled nucleotides. Anti-Cancer Res. 13, 1153–1155.

    Google Scholar 

  42. Zebhe, I., Sällström, J. F., Hacker, G. W., Hauser-Kronberger, C., Rylander, E., and Wilander, E. (1994) Indirect and direct on 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 

  43. Teo, I. A. and Shaunk S. (1995) PCR in situ: an appraisal of an emerging technique. Histochem. J. 27, 647–659.

    PubMed  CAS  Google Scholar 

  44. Bassam et al. (1993) Automated hot start PCR by mineral oil and paraffin wax. Biotechniques 14, 30–34.

    PubMed  CAS  Google Scholar 

  45. Nuovo, G. J., Gallery, F., Hom, R., MacConnell, P., and Bloch, W. (1993) Importance of different variables for enhancing in situ detection of PCR amplified DNA. PCR Meth. Appl. 2, 305–312.

    CAS  Google Scholar 

  46. Myers, T. W. and Gelfand, D. H. (1991) Reverse transcription and DNA amplification by a thermus thermophilus DNA polymerase. Biochemistry 30, 7661–7666.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Pathology In Situ Hybridization Unit, National Cancer Institute National Institutes of Health, Bethesda, MD

    Vishakha Thaker

Authors
  1. Vishakha Thaker
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Catholic University of America, Washington, DC

    Lorette C. Javois

Rights and permissions

Reprints and permissions

Copyright information

© 1999 Humana Press Inc., Totowa, NJ

About this protocol

Cite this protocol

Thaker, V. (1999). In Situ RT-PCR and Hybridization Techniques. In: Javois, L.C. (eds) Immunocytochemical Methods and Protocols. Methods in Molecular Biology™, vol 115. Humana Press. https://doi.org/10.1385/1-59259-213-9:379

Download citation

  • DOI: https://doi.org/10.1385/1-59259-213-9:379

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-813-4

  • Online ISBN: 978-1-59259-213-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation