Methodology for Whole Mount and Fluorescent RNA In Situ Hybridization in Echinoderms: Single, Double, and Beyond

Identifying the location of a specific RNA in a cell, tissue, or embryo is essential to understand its function. Here we use echinoderm embryos to demonstrate the power of fluorescence in situ RNA hybridizations to localize sites of specific RNA accumulation in whole mount embryo applications. We add to this technology the use of various probe-labeling technologies to colabel multiple RNAs in one application and we describe protocols for incorporating immunofluorescence approaches to maximize the information obtained in situ. We offer alternatives for these protocols and troubleshooting advice to identify steps in which the procedure may have failed. Overall, echinoderms are wonderfully suited for these technologies, and these protocols are applicable to a wide range of cells, tissues, and embryos.