Abstract
The isolation and characterization of RNA polymerases from the Salmonella phage SP6 and the E. coli phages T7 and T3 has revolutionized all aspects of the study of RNA metabolism (1–6). Indeed, it is now possible to generate unlimited quantities of virtually any RNA molecule in a chemically pure form. This technology is based on a number of properties of the viral transcription units. First, and in contrast to their cellular counterparts, the enzymes are single-chain proteins that were easily purified from phage-infected cells and are now produced by recombinant DNA technology. Second, they very specifically recognize their own promoters (7 and references therein), which are contiguous 17–20-bp-long sequences rarely encountered in bacterial, plasmid, or eukaryotic sequences. Third, the enzymes are highly processive, allowing the efficient synthesis of very long transcripts from DNA templates. In this chapter, the preparation of the DNA templates, the transcription from the templates of labeled synthetic RNA molecules, commonly called riboprobes, and their use in Northern and RNase protection assays are discussed.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Butler, E. T. and Chamberlin, M. J. (1984) Bacteriophage SP6-specific RNA polymerase. J. Biol. Chem. 257, 5772–5788.
Melton, D. A., Krieg, P. A., Rebagliati, M. R., Maniatis, T., Zinn, K., and Green, M. R. (1984) Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 12, 7035–7056.
Davanloo, P., Rosenberg, A. H., Dunn, J. J., and Studier, F. W. (1984) Cloning and expression of the gene for bacteriophage T7 RNA polymerase. Proc. Natl. Acad. Sci. USA 81, 2035–2039.
Krieg, P. A. and Melton, D. A. (1987) In vitro RNA synthesis with SP6 RNA polymerase. Methods Enzymol. 155, 397–415.
Yisraeli, J. K. and Melton, D. A. (1989) Synthesis of long, capped transcripts in vitro by SP6 and T7 RNA polymerases. Methods Enzymol 180, 42–50.
Milligan, J. F. and Uhlenbeck, O. C. (1989) Synthesis of small RNAs using T7 RNA polymerase. Methods Enzymol. 180, 51–62.
Breaker, R. B., Banerji, A., and Joyce, G. F. (1994) Continuous in vitro evolution of bacteriophage RNA polymerase promoters. Biochemistry 33, 11,980–11,986.
Milligan, J. F., Groebe, D. R., Witherell, G. W., and Uhlenbeck, O. C. (1987) Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucleic Acids Res. 15, 8783–8798.
Roitsch, T. and Lehle, L. (1989) Requirements for efficient in vitro transcription and translation: a study using yeast invertase as a probe. Biochim. Biophys. Acta 1009, 19–26.
Schenbon, E. T. and Mierendorf, R. C. (1985) A novel transcription property of SP6 and T7 RNA polymerases: dependence on template structure. Nucleic Acids Res. 13, 6223–6234.
Nam, S. C. and Kang, C. (1988) Transcription initiation site selection and abortive initiation cycling of phage SP6 RNA polymerase. J. Biol. Chem. 263, 18,123–18,127.
Solazzo, M., Spinelli, L., and Cesareni, G. (1987) SP6 RNA polymerase: sequence requirements downstream from the transcription start site. Focus 10, 11,12.
Stump, W. T. and Hall, K. B. (1993) SP6 RNA polymerase efficiently synthesizes RNA from short double-stranded DNA templates. Nucleic Acids Res. 21, 5480–5484.
Moreau, G. (1991) RNA binding properties of the Xenopus LA proteins. Ph. D. dissertation, University of Geneva, Switzerland.
Taylor, D. R. and Mathews, M. B. (1993) Transcription by SP6 RNA polymerase exhibits an ATP dependence that is influenced by promoter topology. Nucleic Acids Res. 21, 1927–1933.
Sappino, A.-P., Huarte, J., Belin, D., and Vassalli, J.-D. (1989) Plasminogen activators in tissue remodeling and invasion: mRNA localization in mouse ovaries and implanting embryos. J. Cell Biol. 109, 2471–2479.
Jostarndt, K., Puntschart, A., Hoppeler, H., and Billeter, R. (1994) The use of [33P]-labeled riboprobes for in situ hybridizations: localization of myosin light chain mRNAs in adult human skeletal muscle. Histochem. J. 26, 32–40.
Dorries, U., Bartsch, U., Nolte, C., Roth, J., and Schachner, M. (1993) Adaptation of a non-radioactive in situ hybridization method to electron microscopy: detection of tenascin mRNA in mouse cerebellum with digoxigenin-labeled probes and gold-labeled antibodies. Histochemistry 99, 251–262.
Kriegsmann, J., Keyszer, G., Geiler, T., Gay, R. E., and Gay, S. (1994) A new double labeling technique for combined in situ hybridization and immunohistochemical analysis. Lab. Invest. 71, 911–917.
Egger, D., Troxler, M., and Bienz, K. (1994) Light and electron microscopic in situ hybridization: non-radioactive labeling and detection, double hybridization, and combined hybridization-immunocytochemistry. J. Histochem. Cytochem. 42, 815–822.
Pokrovskaya, I. D. and Gurevich, V. V. (1994) In vitro transcription: preparative RNA yields in analytical scale reactions. Anal. Biochem. 220, 420–423.
Krieg, P. A. (1991) Improved synthesis of full length RNA probe at reduced incubation temperatures. Nucleic Acids Res. 18, 6463.
Belin, D., Mudd, E. A., Prentki, P., Yi-Yi, Y., and Krisch, H. M. (1987) Sense and antisense transcription of bacteriophage T4 gene 32. J. Mol. Biol. 194, 231–243.
Mead, D. A., Szesna-Skorupa, E., and Kemper, B. (1986) Single-stranded DNA blue T7 promoter plasmids. Protein Eng. 1, 67–74.
Macdonald, L. E., Durbin, R. K., and McAllister, W. T. (1994) Characterisation of two types of termination signals for bacteriophage T7 RNA polymerase. J. Mol. Biol. 238, 145–158.
Curran, J., Marq, J. B., and Kolakofsky, D. (1992) The Sendai virus nonstructural C proteins specifically inhibit viral mRNA synthesis. Virology 189, 647–656.
Hod, Y. (1992) A simplified ribonuclease protection assay. Biotechniques 13, 852–853.
Lau, E. T., Kong, R. Y. C., and Cheah, K. S. E. (1993) A critical assessment of the RNase protection assay as a means of determining exon sizes. Anal. Biochem. 209, 360–366.
Belin, D., Wohlwend, A., Schleuning, W.-D., Kruithof, E. K. O., and Vassalli, J.-D. (1989) Facultative polypeptide translocation allows a single mRNA to encode the secreted and cytosolic forms of plasminogen activators inhibitor 2. EMBO J. 8, 3287–3294.
Vassalli, J.-D., Huarte, J., Bosco, D., Sappino, A.-P., Sappino, N., Velardi, A., Wohlwend, A., Erno, H., Monard, D., and Belin, D. (1993) Protease-nexin I as an androgen-dependent secretory product of the murine seminal vesicle. EMBO J. 12, 1871–1878.
Lyakhov, D. L., He, B., Zhang, X., Studier, F. W., Dunn, J. J., and McAllister, W. T. (1997) Mutant bacteriophage T7 RNA polymerases with altered termination properties. J. Mol. Biol 269, 28–40.
Scott, P. A. E., Smith, K., Bichmel, R., and Harris, A. L. (1977) Reliable external control for RNase protection assays. Nucleic Acids Res. 95, 1305–1306.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2000 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Belin, D. (2000). RNA Probes for the Analysis of Gene Expression. In: Rapley, R. (eds) The Nucleic Acid Protocols Handbook. Springer Protocols Handbooks. Humana Press, Totowa, NJ. https://doi.org/10.1385/1-59259-038-1:181
Download citation
DOI: https://doi.org/10.1385/1-59259-038-1:181
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-0-89603-459-4
Online ISBN: 978-1-59259-038-4
eBook Packages: Springer Book Archive