Detection and Automated Quantification of Nucleocytoplasmic RNA Fractions in Arabidopsis Using smFISH

Subcellular RNA localization is an underexplored regulatory layer crucial for properly adapting cells to cellular or environmental conditions. Most studies describing RNA localization have been performed by cell fractionation and subsequent RNA quantification from pools of cells, thereby missing information about cell-to-cell variability. RNA single-molecule fluorescent in situ hybridization (smFISH) is an effective technique for detecting single RNA molecules and identifying subcellular accumulation patterns. Nevertheless, obtaining quantitative results from smFISH can be challenging in tissues with high autofluorescence, like in plants. Here, we describe an automated pipeline to detect and quantify nucleocytoplasmic RNA levels from Arabidopsis root smFISH images. This pipeline utilizes free image preprocessing, segmentation, and RNA detection software. The method permits users with any programming skills to analyze batches of images. Suggestions and recommendations for image acquisition, processing, and data analysis are included. This pipeline allows quantitative differences in nucleocytoplasmic distribution at the single-cell level to be studied under different cellular, environmental, and genetic contexts.