RNA Expression Analysis on Formalin-Fixed Paraffin-Embedded Tissues in TMA Format by RNA In Situ Hybridization

  • Jürgen Veeck
  • Edgar DahlEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 664)


The technique of RNA in situ hybridization, i.e., the detection of specific messenger RNA sequences within structurally intact cells or tissues is not widely used in biomedical research, because it can be cumbersome and technically challenging. However, it has a major advantage that warrants and sometimes even requires its application and the associated efforts. RNA in situ hybridization enables a detailed analysis of gene expression in the absence of a suitable antibody to the molecule encoded by the gene of interest. Within the wealth of RNA analysis technologies available nowadays, RNA in situ hybridization still is the only methodology that allows a precise localization of gene expression at a cellular level. This is particularly important if, e.g., new molecular markers or potential drug target molecules have to be analyzed in large cohorts of human tissues. In cancer research, it may be necessary to show that a newly characterized molecule is indeed expressed by the tumor cells themselves, rather than by any surrounding tissue. A protocol is presented here that has been routinely and successfully used on FFPE tissues assembled on a tissue micro array (TMA).

Key words

RNA in situ hybridization messenger RNA (mRNA) Cellular localization Target validation Breast cancer Tumor marker Drug target 



The authors would like to thank the former and present lab technicians Annette Buß, Beate Petschke, and Sevim Alkaya, who constantly helped to further optimize this RNA in situ hybridization protocol over the years.


  1. 1.
    Gall JG, Pardue ML (1969). Formation and detection of RNA-DNA hybrid molecules in cytological preparations. Proc Natl Acad Sci USA;63:378–383.PubMedCrossRefGoogle Scholar
  2. 2.
    John HA, Birnstiel ML, Jones KW (1969). RNA–DNA hybrids at the cytological level. Nature;223:582–587.PubMedCrossRefGoogle Scholar
  3. 3.
    Buongiorno-Nardelli M, Amaldi F (1970). Autoradiographic detection of molecular hybrids between rRNA and DNA in tissue sections. Nature;225:946–947.PubMedCrossRefGoogle Scholar
  4. 4.
    Angerer LM, Angerer RC (1981). Detection of poly A+ RNA in sea urchin eggs and embryos by quantitative in situ hybridization. Nucleic Acids Res;9:2819–2840.PubMedCrossRefGoogle Scholar
  5. 5.
    Miller MA, Kolb PE, Raskind MA (1993). A method for simultaneous detection of multiple mRNAs using digoxigenin and radioisotopic cRNA probes. J Histochem Cytochem;41:1741–1750.PubMedCrossRefGoogle Scholar
  6. 6.
    Pohle T, Shahin M, Gillessen A, Schuppan D, Herbst H, Domschke W (1996). Expression of type I and IV collagen mRNAs in healing gastric ulcers – a comparative analysis using isotopic and non-radioactive in situ hybridization. Histochem Cell Biol;106:413–418.PubMedCrossRefGoogle Scholar
  7. 7.
    Renz M, Kurz C (1984). A colorimetric method for DNA hybridization. Nucleic Acids Res;12:3435–3444.PubMedCrossRefGoogle Scholar
  8. 8.
    Wisden W, Morris B, Hunt S (1991). Molecular Neurobiology, A. Practical Approach (eds. J. Chad and H. Wheal), pp. 205–225. University Press/IRL, Oxford.Google Scholar
  9. 9.
    Dahl E, Winterhager E, Traub O, Willecke K (1995). Expression of gap junction genes, connexin40 and connexin43, during fetal mouse development. Anat Embryol (Berl);191:267–278.CrossRefGoogle Scholar
  10. 10.
    Cox KH, DeLeon DV, Angerer LM, Angerer RC (1984). Detection of mrnas in sea urchin embryos by in situ hybridization using asymmetric RNA probes. Dev Biol;101:485–502.PubMedCrossRefGoogle Scholar
  11. 11.
    Feinberg AP, Vogelstein B (1983). A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem;132:6–13.PubMedCrossRefGoogle Scholar
  12. 12.
    Tautz D, Pfeifle C (1989). A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback. Chromosoma;98:81–85.PubMedCrossRefGoogle Scholar
  13. 13.
    Höltke HJ, Kessler C (1990). Non-radioactive labeling of RNA transcripts in vitro with the hapten digoxigenin (DIG); hybridization and ELISA-based detection. Nucleic Acids Res;18:5843–5851.PubMedCrossRefGoogle Scholar
  14. 14.
    Gilman M (1993). Current Protocols in Molecular Biology (eds. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K), Vol. 1, pp. 4.7.1–4.7.6, Greene and Wiley-Interscience, New York.Google Scholar
  15. 15.
    Zafrakas M, Chorovicer M, Klaman I, Kristiansen G, Wild PJ, Heindrichs U, Knüchel R, Dahl E (2006). Systematic characterisation of GABRP expression in sporadic breast cancer and normal breast tissue. Int J Cancer;118:1453–1459.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  1. 1.Molecular Oncology Group, Institute of PathologyUniversity Hospital of the RWTH AachenAachenGermany

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