LOCALIZATION OF INTRANUCLEAR RNA BY ELECTRON-MICROSCOPY IN-SITU HYBRIDIZATION USING A GENOMIC DNA-PROBE

Background: The presence of RNA in the cell nucleus is well known, How ever, a high resolution in situ hybridization evidence for the presenc e of RNA in some nuclear particles is still lacking, The aim of this w ork is to localize RNA in subnuclear particles using a novel ultrastru ctural in situ hybridization procedure. In this study, biotinylated ge nomic mouse DNA as a probe to localize total RNA in the nuclei of mous e hepatocytes was used. Methods: The procedure is based on paraformald ehyde fixation and embedding in lowicryl resin, Thin sections are moun ted in formvar-coated gold grids. Hybridization is performed on non-de natured thin sections. DNA-RNA hybrids are detected with streptavidin- 10 nm gold particles complex. By controlling the time of nick-translat ion during incorporation of biotin into the probe, labeling in the fib rillar portions of the nucleoplasm is obtained. More digested probes g enerate more labeling in the granular components. Nucleoli were simila rly labeled. Results: As expected, no label was observed in the compac t chromatin clumps. These results indicate that granular components as perichromatin granules in the nucleus contain more processed RNA than fibrillar portions, As a comparison, viral DNA sequences on denatured RNase-treated thin sections of adenovirus-2 (Ad-2)-infected human cel ls were detected. As previously reported, at late stages DNA was obser ved in the viral particles and surrounding nucleoplasm, where Ad-2 DNA is synthesized, Conclusions: The present procedure allows the study o f intranuclear RNA distribution and will be useful for the analysis of RNA processing in several types of cells.