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Localization of intranuclear RNA by electron microscopy in situ hybridization using a genomic DNA probe
G López-Velázquez1, M L Segura-Valdéz, M A Alcántara-Ortigoza
1Laboratorio de Microscopía Electrónica, Facultad de Ciencias, Universidad Nacional Autónoma de México, D.F.
Background:
The presence of RNA in the cell nucleus is well known. However, a high resolution in situ hybridization evidence for the presence of RNA in some nuclear particles is still lacking. The aim of this work is to localize RNA in subnuclear particles using a novel ultrastructural in situ hybridization procedure. In this study, biotinylated genomic mouse DNA as a probe to localize total RNA in the nuclei of mouse hepatocytes was used.
Methods:
The procedure is based on paraformaldehyde fixation and embedding in lowicryl resin. Thin sections are mounted in formvar-coated gold grids. Hybridization is performed on non-denatured thin sections. DNA-RNA hybrids are detected with streptavidin-10 nm gold particles complex. By controlling the time of nick-translation during incorporation of biotin into the probe, labeling in the fibrillar portions of the nucleoplasm is obtained. More digested probes generate more labeling in the granular components. Nucleoli were similarly labeled.
Results:
As expected, no label was observed in the compact 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 cells were detected. As previously reported, at late stages DNA was observed in the viral particles and surrounding nucleoplasm, where Ad-2 DNA is synthesized.
Conclusions:
The present procedure allows the study of intranuclear RNA distribution and will be useful for the analysis of RNA processing in several types of cells.