Spatial mRNA profiling using Rapid Amplified Multiplexed-FISH (RAM-FISH)

Spatial localization of multiple RNA transcripts in intact tissues can reveal gene regulatory interactions that shape development and function. However, multiplex RNA imaging remains challenging to implement in most laboratories because existing techniques often require complex workflows, specialized equipment, or methods that are not optimized for 3D tissue samples. Here, we describe Rapid Amplified Multiplexed Fluorescence In-Situ Hybridization (RAM-FISH), a modular workflow for multiplexed spatial RNA detection in intact tissues, organs, and whole-mount specimens. RAM-FISH achieves multiplexing through iterative cycles of hybridization and imaging, enabling detection of three to four genes per cycle and more than 30 transcripts within the same sample. The workflow is compatible with standard confocal microscopy and performed either manually or using a fluidics automation system. We demonstrate RAM-FISH by visualizing transcription dynamics in developing Bicyclus anynana butterfly wings and intact 14-days-post-fertilization zebrafish larvae. In both, RAM-FISH resolved multi-gene spatial expression patterns in intact tissues that were previously inferred through independent experiments as well as genes characterized for the first time. Together, RAM-FISH offers an accessible open source platform for multiplex spatial transcript detection in diverse experimental systems. Graphical Abstract