Single-molecule RNA Fluorescence in situ Hybridization (smFISH) in Caenorhabditis elegans

Single-molecule RNA fluorescence in situ hybridization (smFISH) is a technique to visualize individual RNA molecules using multiple fluorescently-labeled oligonucleotide probes specific to the target RNA ( Raj et al., 2008 ; Lee et al., 2016a ). We adapted this technique to visualize RNAs in the C. elegans whole adult worm or its germline, which enabled simultaneous recording of nascent transcripts at active transcription sites and mature mRNAs in the cytoplasm ( Lee et al., 2013 and 2016b). Here we describe each step of the smFISH procedure, reagents, and microscope settings optimized for C. elegans extruded gonads.