An Efficient Method to Detect Messenger RNA (mRNA) in the Inner Ear by RNAscope In Situ Hybridization

Abstract

Biological processes are largely governed by the RNA molecules and resulting peptides that are encoded by an organism’s DNA. For decades, our understanding of biology has been vastly enhanced through study of the distribution and abundance of RNA molecules. Studies of the inner ear are no exception, and approaches like qPCR, RNA-seq, and in situ hybridization (ISH) have contributed greatly to our understanding of inner ear development and function. While qPCR and RNA-seq provide sensitive and broad measures of RNA quantity, they can be limited in their ability to resolve RNA localization. Thus, ISH remains a vital technique for inner ear studies. However, traditional ISH approaches can be technically challenging, time-consuming, suffer from high background, and are generally limited to the investigation of only a single RNA of interest. Recent advances in ISH approaches have overcome many of these limitations allowing for speed, high signal-to-noise, and the ability to perform multiplexed ISH where several transcripts of interest can be visualized in the same tissue or section. One such approach is RNAscope which is a commercially available option that allows for ease of use and, for many transcripts, the ability to achieve absolute quantification of RNA molecules per cell. Here we outline RNAscope methods that have been optimized for inner ear (and related) tissues and allow for relatively rapid labeling of RNA transcripts of interest in fixed tissues. Furthermore, these methods elucidate how RNAscope labeling can be imaged with brightfield or fluorescence microscopy, how it allows for quantification as well as localization, how it can be multiplexed to visualize multiple transcripts simultaneously, and how it can be combined with immunocytochemistry so that RNA and proteins may be visualized in the same sample.