Advertisement

Sensitive and Specific In Situ Hybridization for Early Drug Discovery

  • Pernille A. Usher
  • Elisabeth D. Galsgaard
  • Kimberly Kruse
  • Jishu Wang
  • Berit O. Krogh
  • Jette Mandelbaum
  • Kasper AlmholtEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1211)

Abstract

High-throughput analyses of gene expression such as microarrays and RNA-sequencing are widely used in early drug discovery to identify disease-associated genes. To further characterize the expression of selected genes, in situ hybridization (ISH) using RNA probes (riboprobes) is a powerful tool to localize mRNA expression at the cellular level in normal and diseased tissues, especially for novel drug targets, where research tools like specific antibodies are often lacking.

We describe a sensitive ISH protocol using radiolabelled riboprobes suitable for both paraffin-embedded and cryo-preserved tissue. The riboprobes are generated by in vitro transcription using PCR products as templates, which is less time consuming compared to traditional transcription from linearized plasmids, and offers a relatively simple way to generate several probes per gene, e.g., for splice variant analyses. To ensure reliable ISH results, we have incorporated a number of specificity controls in our standard experimental setup. We design antisense probes to cover two non-overlapping parts of the gene of interest, and use the corresponding sense probes as controls for unspecific binding. Probes are furthermore tested on sections of paraffin-embedded or cryo-preserved positive and negative control cells with known gene expression. Our protocol thus provides a method for sensitive and specific ISH, which is suitable for target validation and characterization in early drug discovery.

Key words

In situ hybridization Drug discovery Gene expression Radiolabelled riboprobe Non-overlapping Paraffin-embedded Cryo-preserved Control cells Splice variants 

References

  1. 1.
    Cox KH, DeLeon DV, Angerer LM et al (1984) Detection of mRNAs in sea urchin embryos by in situ hybridization using asymmetric RNA probes. Dev Biol 101:485–502PubMedCrossRefGoogle Scholar
  2. 2.
    Holland PW, Harper SJ, McVey JH et al (1987) In vivo expression of mRNA for the Ca++-binding protein SPARC (osteonectin) revealed by in situ hybridization. J Cell Biol 105:473–482PubMedCrossRefGoogle Scholar
  3. 3.
    Kristensen P, Eriksen J, Danø K (1991) Localization of urokinase-type plasminogen activator messenger RNA in the normal mouse by in situ hybridization. J Histochem Cytochem 39:341–349PubMedCrossRefGoogle Scholar
  4. 4.
    Rømer J, Hasselager E, Nørby PL et al (2003) Epidermal overexpression of interleukin-19 and -20 mRNA in psoriatic skin disappears after short-term treatment with cyclosporine A or calcipotriol. J Invest Dermatol 121:1306–1311PubMedCrossRefGoogle Scholar
  5. 5.
    David R, Wedlich D (2001) PCR-based RNA probes: a quick and sensitive method to improve whole mount embryo in situ hybridizations. Biotechniques 30(769–772):774Google Scholar
  6. 6.
    Chomczynski P (1992) Solubilization in formamide protects RNA from degradation. Nucleic Acids Res 20:3791–3792PubMedCrossRefPubMedCentralGoogle Scholar
  7. 7.
    Poulsom R, Longcroft JM, Jeffery RE et al (1998) A robust method for isotopic riboprobe in situ hybridisation to localise mRNAs in routine pathology specimens. Eur J Histochem 42:121–132PubMedGoogle Scholar
  8. 8.
    Stevens R, Stevens L, Price N (1983) The stabilities of various thiol compounds used in protein purifications. Biochem Edu 11:70CrossRefGoogle Scholar
  9. 9.
    Durocher Y, Perret S, Kamen A (2002) High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells. Nucleic Acids Res 30:E9PubMedCrossRefPubMedCentralGoogle Scholar
  10. 10.
    Wilcox JN (1993) Fundamental principles of in situ hybridization. J Histochem Cytochem 41:1725–1733PubMedCrossRefGoogle Scholar
  11. 11.
    Lisowski AR, English ML, Opsahl AC et al (2001) Effect of the storage period of paraffin sections on the detection of mRNAs by in situ hybridization. J Histochem Cytochem 49:927–928PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Pernille A. Usher
    • 1
  • Elisabeth D. Galsgaard
    • 1
  • Kimberly Kruse
    • 2
  • Jishu Wang
    • 3
  • Berit O. Krogh
    • 1
  • Jette Mandelbaum
    • 1
  • Kasper Almholt
    • 1
    Email author
  1. 1.Biopharmaceuticals Research UnitNovo Nordisk A/SMåløvDenmark
  2. 2.Biopharmaceuticals Research UnitNovo Nordisk Inc.SeattleUSA
  3. 3.Biopharmaceuticals Research UnitNovo Nordisk China R&DBeijingChina

Personalised recommendations