A technical review and guide to RNA fluorescence in situ hybridization

Abstract

RNA-fluorescence in situ hybridization (FISH) is a powerful tool to visualize target messenger RNA transcripts in cultured cells, tissue sections or whole-mount preparations. As the technique has been developed over time, an ever-increasing number of divergent protocols have been published. There is now a broad selection of options available to facilitate proper tissue preparation, hybridization, and post-hybridization background removal to achieve optimal results. Here we review the technical aspects of RNA-FISH, examining the most common methods associated with different sample types including cytological preparations and whole-mounts. We discuss the application of commonly used reagents for tissue preparation, hybridization, and post-hybridization washing and provide explanations of the functional roles for each reagent. We also discuss the available probe types and necessary controls to accurately visualize gene expression. Finally, we review the most recent advances in FISH technology that facilitate both highly multiplexed experiments and signal amplification for individual targets. Taken together, this information will guide the methods development process for investigators that seek to perform FISH in organisms that lack documented or optimized protocols.

Keywords: FISH; Hybridization; Oligonucleotide probe; Protocol development; Riboprobe; Whole mount; mRNA expression.

Figure 1. Schematic representation of the technical development of fluorescent in situ hybridization (FISH).

In situ hybridization (ISH) was first performed by Gall & Pardue (1969) using radioactive probes. Fluorescent ISH (FISH) against DNA was first performed by Rudkin & Stollar (1977). FISH against RNA (RNA-FISH) was first performed by Singer & Ward (1982). RNA-FISH that could be used to resolve individual mRNA transcripts was first performed by Femino et al. (1998) and later improved upon in whole mount tissue by Raj et al. (2008). Horseradish peroxidase-based chromogenic (or colorimetric) ISH was later introduced by Tanner et al. (2000) as an alternative FISH without the need for a fluorescence microscope.

Figure 2. Schematic representation of the riboprobe and oligonucleotide in situ hybridization probe types.

(A) Hapten-labeled RNA probes must be bound by an antibody labeled with a fluorophore to allow for visualization. (B) DNA oligomers directly labeled with a fluorophore can be directly visualized.