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
Part of the Methods in Molecular Biology book series (MIMB, volume 1211)


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 


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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

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