Locating RNAs in situ with FISH-STIC probes

The location of a molecule within the cell often provides important clues to its function and regulation, therefore techniques to locate RNA within cells are vital tools to study noncoding RNA function. Fluorescence in situ hybridization (FISH) is a simple and reliable approach to locate RNAs in any cell type. Intracellular localization of RNA using FISH (RNA-FISH) requires resolution at the single cell and single molecule level which can be achieved using fluorescent-labeled nucleic acid antisense probes. Sequential Tagged and Intertwined oligodeoxyribonucleotide Complex (FISH-STIC) probes are a straightforward means for laboratories to design their own FISH probes that can be synthesized commercially. Here we provide a detailed protocol for applying FISH-STIC probes for in situ hybridization on cultured cells as a convenient and flexible method for localizing individual RNAs with many fluorophores using fluorescence microscopy.