Production of Digoxigenin-Labeled Riboprobes for In Situ Hybridization Experiments.

Kristen S Barratt, Ruth M Arkell

Current protocols in mouse biology 2020 Jun

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Experiments that visualize gene expression in intact tissues or organisms are fundamental to studies of gene function. These experiments, called in situ hybridization, require the production of a riboprobe, which is a labeled antisense RNA corresponding to a particular gene. The most commonly used system for visualizing gene expression via in situ hybridization is the incorporation of a digoxigenin label into an in vitro-transcribed RNA probe. After hybridization of the riboprobe to a target mRNA, its location can be detected via a high-affinity α-digoxigenin antibody conjugated to an alkaline-phosphatase enzyme. The article describes the design and production of digoxigenin-labeled riboprobes transcribed in vitro from template DNA (either plasmid or PCR amplicon). These riboprobes are suitable for use in tissue and whole-mount in situ hybridization protocols. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Plasmid-derived riboprobes Alternate Protocol: PCR-derived riboprobes Basic Protocol 2: Riboprobe synthesis with DIG label. © 2020 John Wiley & Sons, Inc.

Citation

Kristen S Barratt, Ruth M Arkell. Production of Digoxigenin-Labeled Riboprobes for In Situ Hybridization Experiments. Current protocols in mouse biology. 2020 Jun;10(2):e74