Whole Mount In Situ Hybridization (WM-ISH) for miRNA Expression Profiling During Vertebrate Development

Abstract

Knowledge of tissue-specific and cell-specific expression patterns of miRNAs can directly inform functional studies. However, detailed analysis of spatial patterns of miRNA expression has been technically challenging. While the regular ISH technique has been extensively used for characterizing cellular localization and tissue distribution of miRNAs in tissue and cell preparations, it cannot be efficiently applied to monitor miRNA expression in whole animals. Utilization of locked nucleic acids (LNAs) in ISH, however, has allowed for the whole mount in situ hybridization techniques (WM-ISH) to monitor miRNA expression in whole animals or animal embryos. The WM-ISH techniques have been tested in Drosophila, zebrafish, chicken, and mouse embryos by several laboratories (Proc Natl Acad Sci USA 102:18017–18022, 2005; Science 309:310–311, 2005; Nat Methods 3:27–29, 2006; Dev Dyn 235:3156–3165, 2006). These studies led us to tremendous insight into the spatial and temporal patterns of miRNA expression and control of embryonic development by miRNAs. The major features and advantages of the WM-ISH techniques are whole mount analysis and high-throughput profiling of miRNAs. The data from WM-ISH highlight cell-type, organ or structure-specific expression, localization within germ layers and their derivatives, and expression in multiple cell and tissue types and within subregions of structures and tissues, the information which is otherwise inaccessible with other miRNA expression detection methods. This chapter mainly introduces the methods provided in the study by Darnell et al. (Dev Dyn 235:3156–3165, 2006) from the Department of Cell Biology and Anatomy, University of Arizona (Tucson, Arizona, USA).