title: Sensitive and specific in situ hybridization for early drug discovery.
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.
authors.affiliation: Biopharmaceuticals Research Unit, Novo Nordisk A/S, Novo Nordisk Park, 2760, Måløv, Denmark.
authors.forename: Pernille A
authors.initials: PA
authors.lastname: Usher
authors.forename: Elisabeth D
authors.initials: ED
authors.lastname: Galsgaard
authors.forename: Kimberly
authors.initials: K
authors.lastname: Kruse
authors.forename: Jishu
authors.initials: J
authors.lastname: Wang
authors.forename: Berit O
authors.initials: BO
authors.lastname: Krogh
authors.forename: Jette
authors.initials: J
authors.lastname: Mandelbaum
authors.forename: Kasper
authors.initials: K
authors.lastname: Almholt
mesh_terms: D000818:Animals; D015925:Cryopreservation / Q000379:methods; D055808:Drug Discovery / Q000379:methods; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods*; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods; D015347:RNA Probes / Q000032:analysis / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D014158:Transcription, Genetic
chemical_list: D015347:RNA Probes; D012333:RNA, Messenger