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) sequen ces in fixed cells. This technique does not, however, provide dynamic information on the intracellular behavior of the targeted molecules. T he aim of this work was to investigate possibilities of labeled DNA pr obes for RNA detection in cells that are maintained alive. Such techni ques will provide useful tools for studying dynamic cellular processes such as RNA distribution and transport from transcription sites to tr anslation sites by means of fluorescence microscopy. First a reversibl e, nonperturbing cell permeabilization procedure was developed using s treptolysin O. This procedure was used to introduce oligodeoxynucleoti des 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 gr owth perturbation was observed after permeabilization, (ii) introducti on of a 28S RNA-specific probe (plasmid and oligonucleotides) into liv ing cells led to bright nucleoli and a low cytoplasmic signal, and (ii i) negative control probes did not lead to any fluorescent staining. T hese results indicate that specific hybridization of labeled nucleic a cid probes takes place while cells are maintained under normal physiol ogical conditions. (C) 1997 Academic Press.