An easy to use tool for the analysis of subcellular mRNA transcript colocalisation in smFISH data

Bentley-Abbot, Calum and Heslop, Rhiannon and Pirillo, Chiara and Chandrasegaran, Praveena and McConnell, Gail and Roberts, Ed and Hutchinson, Edward and MacLeod, Annette (2024) An easy to use tool for the analysis of subcellular mRNA transcript colocalisation in smFISH data. Scientific Reports, 14 (1). 8348. ISSN 2045-2322 (

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Single molecule fluorescence in situ hybridisation (smFISH) has become a valuable tool to investigate the mRNA expression of single cells. However, it requires a considerable amount of programming expertise to use currently available open-source analytical software packages to extract and analyse quantitative data about transcript expression. Here, we present FISHtoFigure, a new software tool developed specifically for the analysis of mRNA abundance and co-expression in QuPath-quantified, multi-labelled smFISH data. FISHtoFigure facilitates the automated spatial analysis of transcripts of interest, allowing users to analyse populations of cells positive for specific combinations of mRNA targets without the need for computational image analysis expertise. As a proof of concept and to demonstrate the capabilities of this new research tool, we have validated FISHtoFigure in multiple biological systems. We used FISHtoFigure to identify an upregulation in the expression of Cd4 by T-cells in the spleens of mice infected with influenza A virus, before analysing more complex data showing crosstalk between microglia and regulatory B-cells in the brains of mice infected with Trypanosoma brucei brucei. These analyses demonstrate the ease of analysing cell expression profiles using FISHtoFigure and the value of this new tool in the field of smFISH data analysis.