In Situ Hybridization for Detecting Mature MicroRNAs In Vivo at Single-Cell Resolution

Abstract

MicroRNAs (miRNAs) are key regulators of cell and tissue development. However, spatial resolution of miRNA heterogeneity and accumulation patterns in vivo remains uncharted. Next-generation sequencing methods assay miRNA abundance in tissues, yet these analyses do not provide spatial resolution. A method to assay miRNA expression at single-cell resolution in vivo should clarify the cell-autonomous functions of miRNAs, their roles in influencing the cellular microenvironment, and their perdurance and turnover rate. We present an in situ hybridization protocol to map miRNA subcellular expression in single cells in vivo in four days. Using this protocol, we mapped distinct miRNAs that accumulate in the cytoplasm of one sibling oocyte but not another, dependent on the oocyte developmental stage. Thus, this method provides spatial and temporal resolution of the heterogeneity in expression of miRNAs during Caenorhabditis elegans oogenesis. This protocol can generally be adapted to any tissue amenable to dissection and fixation. © 2019 by John Wiley & Sons, Inc.