Abstract
The characterization of alternatively spliced RNA is a frequently performed task in the molecular biology laboratory. Several methods have been established to characterize specific transcripts, of which microarrays, northern analysis, RT-PCR and nuclease protection assays are the most frequently performed methods in the laboratory. Here, we describe the analysis of alternatively spliced RNA by using 5′-end labelled DNA oligonucleotide probes and S1 nuclease. The method is sensitive, allowing detection of as little as a few hundred femtograms of a specific RNA, and useful for the quantitation of alternatively spliced mRNA isoforms. Because of its insensitivity towards RNA secondary structures and partially degraded RNA, it may perform better in the quantitation of RNA than northern analysis or RT-PCR, especially when long transcripts are studied.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Venter, J. C., Adams, M. D., Myers, E. W., Li, P. W., Mural, R. J., Sutton, G. G., Smith, H. O., Yandell, M., Evans, C. A., Holt, R. A., Gocayne, J. D., Amanatides, P., Ballew, R. M., Huson, D. H., Wortman, J. R., Zhang, Q., Kodira, C. D., Zheng, X. H., Chen, L., Skupski, M., Subramanian, G., Thomas, P. D., Zhang, J., Gabor Miklos, G. L., Nelson, C., Broder, S., Clark, A. G., Nadeau, J., McKusick, V. A., Zinder, N., Levine, A. J., Roberts, R. J., Simon, M., Slayman, C., Hunkapiller, M., Bolanos, R., Delcher, A., Dew, I., Fasulo, D., Flanigan, M., Florea, L., Halpern, A., Hannenhalli, S., Kravitz, S., Levy, S., Mobarry, C., Reinert, K., Remington, K., Abu-Threideh, J., Beasley, E., Biddick, K., Bonazzi, V., Brandon, R., Cargill, M., Chandramouliswaran, I., Charlab, R., Chaturvedi, K., Deng, Z., Di Francesco, V., Dunn, P., Eilbeck, K., Evangelista, C., Gabrielian, A. E., Gan, W., Ge, W., Gong, F., Gu, Z., Guan, P., Heiman, T. J., Higgins, M. E., Ji, R. R., Ke, Z., Ketchum, K. A., Lai, Z., Lei, Y., Li, Z., Li, J., Liang, Y., Lin, X., Lu, F., Merkulov, G. V., Milshina, N., Moore, H. M., Naik, A. K., Narayan, V. A., Neelam, B., Nusskern, D., Rusch, D. B., Salzberg, S., Shao, W., Shue, B., Sun, J., Wang, Z., Wang, A., Wang, X., Wang, J., Wei, M., Wides, R., Xiao, C., Yan, C., et al. (2001) The sequence of the human genome. Science 291, 1304–1351.
Schmucker, D., Clemens, J. C., Shu, H., Worby, C. A., Xiao, J., Muda, M., Dixon, J. E., Zipursky, S. L. (2000) Drosophila Dscam is an axon guidance receptor exhibiting extraordinary molecular diversity. Cell 101, 671–684.
Srinivasan, K., Shiue, L., Hayes, J. D., Centers, R., Fitzwater, S., Loewen, R., Edmondson, L. R., Bryant, J., Smith, M., Rommelfanger, C., Welch, V., Clark, T. A., Sugnet, C. W., Howe, K. J., Mandel-Gutfreund, Y., Ares, M., Jr. (2005) Detection and measurement of alternative splicing using splicing-sensitive microarrays. Methods 37, 345–359.
Alwine, J. C., Kemp, D. J., Parker, B. A., Reiser, J., Renart, J., Stark, G. R., Wahl, G. M. (1979) Detection of specific RNAs or specific fragments of DNA by fractionation in gels and transfer to diazobenzyloxymethyl paper. Methods Enzymol 68, 220–242.
Alwine, J. C., Kemp, D. J., Stark, G. R. (1977) Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc Natl Acad Sci USA 74, 5350–5354.
Mullis, K., Faloona, F., Scharf, S., Saiki, R., Horn, G., Erlich, H. (1986) Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harb Symp Quant Biol 51(Pt 1), 263–273.
Mullis, K. B., Faloona, F. A. (1987) Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol 155, 335–350.
Saiki, R. K., Scharf, S., Faloona, F., Mullis, K. B., Horn, G. T., Erlich, H. A., Arnheim, N. (1985) Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230, 1350–1354.
Gilman, M. (ed.) (2001) Ribonuclease protection assay, in (Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. A., eds.), Current Protocols in Molecular Biology. Wiley, New York, NY, 4.7.1–4.7.8.
Quarless, S. A., Heinrich, G. (1986) The use of complementary RNA and S1 nuclease for the detection and quantitation of low abundance mRNA transcripts. BioTechniques 4, 434–438.
Casey, J., Davidson, N. (1977) Rates of formation and thermal stabilities of RNA:DNA and DNA:DNA duplexes at high concentrations of formamide. Nucleic Acids Res 4, 1539–1552.
Ando, T. (1966) A nuclease specific for heat-denatured DNA in isolated from a product of Aspergillus oryzae. Biochim Biophys Acta 114, 158–168.
Samanta, H. K., Engel, D. (1987) Deionization of formamide with Biorad AG501-X(D). J Biochem Biophys Methods 14, 261–266.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Lützelberger, M., Kjems, J. (2011). S1 Nuclease Analysis of Alternatively Spliced mRNA. In: Nielsen, H. (eds) RNA. Methods in Molecular Biology, vol 703. Humana Press. https://doi.org/10.1007/978-1-59745-248-9_11
Download citation
DOI: https://doi.org/10.1007/978-1-59745-248-9_11
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-58829-913-0
Online ISBN: 978-1-59745-248-9
eBook Packages: Springer Protocols