Detection of Sites of RNA Synthesis

Abstract

The statement made for the visualization of DNA also fully applies to that of RNA: it can be readily detected by immunoelectron microscopy with high precision and specificity by labelling of ultrathin sections from cells and tissues. Antibodies against RNA can be applied, but the reagent most often used is the nucleotide analog 5′bromouridine-5-triphosphate (BrUTP), which, after its incorporation into RNA, can be detected with the monoclonal anti-bromodeoxyuridine antibody and gold-labelled secondary antibody with high spatial resolution. Formaldehyde or formaldehyde/glutaraldehyde fixation combined with embedding in Epon, Lowicryl K4M or LR white resins provides high detection sensitivity. The post-embedding immunogold labelling can be combined with other histochemical stains. Since 5′bromouridine-5-triphosphate is not membrane permeable, permeabilized cells were used or the nucleotide analog was microinjected into cells. Alternative approaches have been the use of the nucleoside analog BrU rather than BrUTP and of liposome transfection vectors.