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 gene-regulatory interactions underlying biological function. Existing technologies for multiplexed RNA localization are expensive, computationally and experimentally complex, have elaborate sample preparation steps, have thin-slice limitations, and require weeks of processing time. This limits the routine use of such techniques in most labs. Here, we describe an easy-to-use methodology, Rapid Amplified Multiplexed-FISH (or RAM-FISH), for localizing dozens of transcripts in the same sample. This methodology achieves multiplexing by localizing 3-4 genes per cycle to detect 30 or more genes. All steps can be done manually or with the help of automation. The method can be applied to fixed tissue sections, entire organs, or whole organisms such as larval Danio rerio , without extensive sample preparation steps. 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 14-days post fertilization zebrafish larvae.