Single-molecule RNA FISH, also known as Stellaris® RNA FISH or smFISH, is a method of detecting and quantifying mRNA and other long RNA molecules in a thin layer of tissue sample. Targets can be reliably imaged through the application of multiple short singly labeled oligonucleotide probes. The binding of up to 48 fluorescent labeled oligos to a single molecule of mRNA provides sufficient fluorescence to accurately detect and localize each target mRNA in a wide-field fluorescent microscopy image. Probes not binding to the intended sequence do not achieve sufficient localized fluorescence to be distinguished from background.
Single-molecule RNA FISH assays can be performed in simplex or multiplex, and can be used as a follow-up experiment to quantitative PCR, or imaged simultaneously with a fluorescent antibody assay. The technology has potential applications in cancer diagnosis, neuroscience, gene expression analysis, and companion diagnostics.