Imaging individual mRNA molecules using multiple singly labeled probes

A strategy using 48 or more singly labeled fluorescent oligonucleotide probes targeted to individual mRNA molecules allows the simultaneous localization and quantification of three mRNA species in fixed cells. mRNA visualization in whole animals and other organisms is also demonstrated. We describe a method for imaging individual mRNA molecules in fixed cells by probing each mRNA species with 48 or more short, singly labeled oligonucleotide probes. This makes each mRNA molecule visible as a computationally identifiable fluorescent spot by fluorescence microscopy. We demonstrate simultaneous detection of three mRNA species in single cells and mRNA detection in yeast, nematodes, fruit fly wing discs, and mammalian cell lines and neurons.