Summary
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.
Similar content being viewed by others
References
Bishop, J.O., Rosbash, M., Evans, D.: Polynucleotide sequences in eukaryotic DNA and RNA that form ribonuclease-resistant complexes with polyuridylic acid. J. Mol. Biol. 85, 75–86 (1974)
Blobel, G.: A protein of molecular weight 78,000 bound to the polyadenylate region of eukaryotic messenger RNAs. Proc. Natl. Acad. Sci. USA 70, 924–928 (1973)
Brahic, M., Haase, A.T.: Detection of viral sequences of low reiteration frequency by in situ hybridization. Proc. Natl. Acad. Sci. USA 75, 6125–6129 (1978)
Conkie, D., Affara, N., Harrison, P.R., Paul, J., Jones, K.W.: In situ localization of globin messenger RNA formation. II. After treatment of Friend virus-transformed mouse cells with dimethyl sulfoxide. J. Cell Biol. 63, 414–419 (1974)
Godard, C.M., Jones, K.W.: Detection of AKR MuLV-specific RNA in AKR mouse cells by in situ hybridization. Nucleic Acids Research 6, 2849–2861 (1979)
Godard, C.M.: Improved method for detection of transcripts by in situ hybridization: detection of virus-specific RNA in avian cells producing Rous sarcoma virus (Manuscript in preparation)
John, H.A., Patrinou-Georgoulas, M., Jones, K.W.: Detection of myosin heavy chain mRNA during myogenesis in tissue culture by in vitro and in situ hybridization. Cell 12, 501–508 (1977)
Jones, K.W., Bishop, J.O., Brito-da-Cunha, A.: Complex formation between poly-r(U) and various chromosomal loci in Rhynchosciara. Chromosoma 43, 375–390 (1973)
Kwan, S.W., Brawerman, G.: A particle associated with the polyadenylate segment in mammalian messenger RNA. Proc. Natl. Acad. Sci. USA 69, 3247–3250 (1972)
Minty, A.J., Birnie, G.D., Paul, J.: Gene expression in Friend erythroleukemia cells following the induction of hemoglobin synthesis. Exp. Cell Res. 115, 1–14 (1978)
Moar, M.H., Jones, K.W.: Detection of virus-specific DNA and RNA base-sequences in individual cells transformed or infected by adenovirus type 2. Int. J. Cancer 16, 998–1007 (1975)
Riley, M., Maling, B., Chamberlin, M.J.: Physical and chemical characterization or two-and three-stranded adenine-thymine and adenine-uracil homopolymer complexes. J. Mol. Biol. 20, 359–389 (1966)
Rosbash, M., Ford, P.J.: Polyadenylic acid-containing RNA in Xenopus laevis oocytes. J. Mol. Biol. 85, 87–101 (1974)
Stevens, C., Felsenfeld, G.: The conversion of two-stranded poly(A+U) to three-strand poly(A+2U) and poly(A) by heat. Biopolymers 2, 293–314 (1964)
Szabo, P., Elder, R., Steffensen, D.M., Uhlenbeck, O.C.: Quantitative in situ hybridization of ribosomal RNA species to polytene chromosomes of Drosophila melanogaster. J. Mol. Biol. 115, 539–563 (1977)
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Godard, C.M., Jones, K.W. Improved method for detection of cellular transcripts by in situ hybridization: Detection of poly (A) sequences in individual cells. Histochemistry 65, 291–300 (1980). https://doi.org/10.1007/BF00493178
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00493178