Single-Molecule Fluorescence In Situ Hybridization (FISH) of Circular RNA CDR1as

Methods Mol Biol. 2018;1724:77-96. doi: 10.1007/978-1-4939-7562-4_7.


Individual mRNA molecules can be imaged in fixed cells by hybridization with multiple, singly labeled oligonucleotide probes, followed by computational identification of fluorescent signals. This approach, called single-molecule RNA fluorescence in situ hybridization (smRNA FISH), allows subcellular localization and absolute quantification of RNA molecules in individual cells. Here, we describe a simple smRNA FISH protocol for two-color imaging of a circular RNA, CDR1as, simultaneously with an unrelated messenger RNA. The protocol can be adapted to circRNAs that coexist with overlapping, noncircular mRNA isoforms produced from the same genetic locus.

Keywords: Absolute quantification of gene expression; FISH; In situ hybridization; Single-molecule detection; circRNA quantification; circRNA visualization.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Gene Expression Regulation*
  • Humans
  • In Situ Hybridization, Fluorescence / methods*
  • RNA / genetics*
  • RNA, Circular
  • RNA, Long Noncoding / genetics*
  • Single-Cell Analysis / methods*


  • RNA, Circular
  • RNA, Long Noncoding
  • long non-coding RNA CDR1AS, human
  • RNA