In situ hybridization in living cells: detection of RNA molecules

Fluorescence hybridization is a widely used technique in cell biology and pathology for detecting specific nucleic acid (DNA and RNA) sequences in fixed cells. This technique does not, however, provide dynamic information on the intracellular behavior of the targeted molecules. The aim of this work was to investigate possibilities of labeled DNA probes for RNA detection in cells that are maintained alive. Such techniques will provide useful tools for studying dynamic cellular processes such as RNA distribution and transport from transcription sites to translation sites by means of fluorescence microscopy. First a reversible, nonperturbing cell permeabilization procedure was developed using streptolysin O. This procedure was used to introduce oligodeoxynucleotides and fluorochrome-labeled DNA probes specific for 28S ribosomal RNA (2.1 kb) into living cells, which were then analyzed by fluorescence microscopy. The results showed that: (i) no increased cell death or growth perturbation was observed after permeabilization, (ii) introduction of a 28S RNA-specific probe (plasmid and oligonucleotides) into living cells led to bright nucleoli and a low cytoplasmic signal, and (iii) negative control probes did not lead to any fluorescent staining. These results indicate that specific hybridization of labeled nucleic acid probes takes place while cells are maintained under normal physiological conditions.