A spatial mRNA profiling workflow using Rapid Amplified Multiplex FISH (RAMFISH)

Spatial localization of multiple mRNAs in intact tissues provides vital insights into development and function, yet routine multiplexed imaging remains constrained by complex workflows, specialized equipment costs, or sample preparation requirements. Here, we present Rapid Amplified Multiplexed FISH (RAMFISH), an accessible, modular, benchtop workflow for targeted spatial mRNA profiling in intact tissues and whole-mount specimens. RAMFISH achieves reliable multiplexing of over 30 transcripts through iterative cycles of standard hybridization either manually or via open-source automation and imaging in shared confocal systems. To streamline analysis, we introduce an integrated, open-source pipeline automating signal normalization, rigid and non-rigid alignment, Laplacian of Gaussian spot calling, and 30-channel composite merging. We validate the platform by mapping multiple transcripts in developing Bicyclus anynana butterfly wings and intact 14 days-post-fertilization Danio rerio larvae, resolving both historically inferred domains and newly characterized gene expression. Overall, RAMFISH methodology offers an end-to-end, completely open-source ecosystem for robust, semi-quantitative, spatial mRNA mapping on a variety of sample types. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/627193v4_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@14dc9a3org.highwire.dtl.DTLVardef@1f11e3dorg.highwire.dtl.DTLVardef@f85483org.highwire.dtl.DTLVardef@1746701_HPS_FORMAT_FIGEXP M_FIG C_FIG