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

Localizing multiple RNA molecules simultaneously in intact tissues and organs is valuable for gaining insights into possible gene-regulatory interactions underlying cell differentiation. Existing technologies for multiplexed RNA localization are expensive, computationally complex, have elaborate sample preparation steps, have size limitations, and require weeks of processing time. This limits the widespread use of such techniques in most labs. Here we describe a cost-effective methodology, Rapid Amplified Multiplexed-FISH (or RAM-FISH), based on Hybridization Chain Reaction 3.0 for localizing dozens of transcripts in the same sample. This methodology achieves multiplexing by localizing 3 genes per cycle to detect 30 or more genes within a few days. The method can be applied to fixed tissue sections, entire organs, or whole organisms such as larval Danio rerio , without extensive sample preparation steps. The automation used here can also be adapted to perform other amplification-based FISH. Here, we demonstrate its utility, flexibility, and versatility for gene expression analysis in two very different types of samples, Bicyclus anynana butterfly larval wings and intact 10-day-old Danio rerio fish larvae.