Design, Labeling, and Application of Probes for RNA smFISH

Methods Mol Biol. 2022;2537:173-183. doi: 10.1007/978-1-0716-2521-7_10.


Visualization of single mRNA molecules in fixed cells can be achieved using single molecule fluorescent in situ hybridization (smFISH). This approach enables accurate quantification of mRNA numbers and localization at a single-cell level. To ensure reliable results using smFISH, it is critical to use fluorescent probes that are highly specific to their RNA target. To facilitate probe design, we have created anglerFISH, a user-friendly command-line based pipeline. In this chapter, we present how to perform a smFISH experiment using user-designed and labeled probes.

Keywords: Command line interface (CLI); FISH probe design; Fluorescence labeling of oligonucleotides; Nextflow workflow; Single-molecule fluorescence in situ hybridization (smFISH).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Fluorescent Dyes*
  • In Situ Hybridization, Fluorescence / methods
  • Nanotechnology
  • Oligonucleotide Probes / genetics
  • RNA* / genetics
  • RNA, Messenger / genetics


  • Fluorescent Dyes
  • Oligonucleotide Probes
  • RNA, Messenger
  • RNA