Improved method for detection of cellular transcripts by in situ hybridization: detection of poly (A) sequences in...

A method for detecting RNA base sequences in individual cells by in situ hybridization with radioactive probes and autoradiography was developed. Cells are fixed with glutaraldehyde at low concentration and deproteinization of fixed target molecules with acid is omitted. Hybridization is carried out in acetate buffer, at low pH, and unhybridized molecules are digested with enzymes prior to autoradiographic exposure. In order to test the specificity of the autoradiographic labelling obtained under these conditions, 3H-poly(U) was hybridized to poly (A) sequences from fixed cells and in vitro and in situ hybridization were compared. It was found that the hybridization reaction of poly(U) in situ obeyed the expectations for such a reaction in solution in that it occurred under the same conditions of ionic strength and temperature, exhibited fast kinetics, was reversed by heat and was sensitive to competition from cold poly(U) but not from other macromolecules. On the other hand, some factors which promote artefactual attachment of probes were identified.