Transcript Localization in Dictyostelium discoideum Cells by RNA FISH

Biogenesis of ribosomal RNA (rRNA) takes place preliminary in the nucleolus of eukaryotic cells, the site of rDNA transcription. Several processing steps of rRNA molecules have been implied to take place in the cytoplasm. To follow these processing events we have adapted protocols for fluorescence in situ hybridization (FISH) for use in Dictyostelium discoideum . We describe methods for the generation of suitable fluorescently labeled probes and the fixation of cells, by which we have localized different precursor and mature rRNA molecules to the nucleolus or the cytoplasm, respectively.