Interrogating RNA and protein spatial subcellular distribution in smFISH data with DypFISH

Anca F. Savulescu*, Robyn Brackin, Emmanuel Bouilhol, Benjamin Dartigues, Jonathan H. Warrell, Mafalda R. Pimentel, Nicolas Beaume, Isabela C. Fortunato, Stephane Dallongeville, Mikaël Boulle, Hayssam Soueidan, Fabrice Agou, Jan Schmoranzer, Jean Christophe Olivo-Marin, Cláudio A. Franco, Edgar R. Gomes, Macha Nikolski, Musa M. Mhlanga

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

8 Citations (Scopus)


Advances in single-cell RNA sequencing have allowed for the identification of cellular subtypes on the basis of quantification of the number of transcripts in each cell. However, cells might also differ in the spatial distribution of molecules, including RNAs. Here, we present DypFISH, an approach to quantitatively investigate the subcellular localization of RNA and protein. We introduce a range of analytical techniques to interrogate single-molecule RNA fluorescence in situ hybridization (smFISH) data in combination with protein immunolabeling. DypFISH is suited to study patterns of clustering of molecules, the association of mRNA-protein subcellular localization with microtubule organizing center orientation, and interdependence of mRNA-protein spatial distributions. We showcase how our analytical tools can achieve biological insights by utilizing cell micropatterning to constrain cellular architecture, which leads to reduction in subcellular mRNA distribution variation, allowing for the characterization of their localization patterns. Furthermore, we show that our method can be applied to physiological systems such as skeletal muscle fibers.

Original languageEnglish
Article number100068
JournalCell Reports Methods
Issue number5
Publication statusPublished - 27 Sept 2021
Externally publishedYes


  • Image analysis
  • Microfabricated patterns
  • Ripley's K
  • RNA subcellular localization
  • Single-molecule FISH


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