Summary
Sequence elements within mRNA-untranslated regions and their binding partners are key controllers of mRNA stability. Changes in mRNA stability can often be detected by changes in steady-state mRNA abundance, or a more careful analysis of mRNA half-lives can be performed following transcriptional repression. This chapter presents methods to isolate RNA from both yeast and mammalian cells for either steady-state or half-life analyses. In addition, two reliable methods to quantitate mRNA levels, northern blot analysis and real-time PCR, are outlined and compared.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Derrigo, M., Cestelli, A., Savettieri, G., and Di Liegro, I. (2000) RNA-protein interactions in the control of stability and localization of messenger RNA. Int. J. Mol. Med. 5, 111–123.
Grzybowska, E. A., Wilczynska, A., and Siedlecki, J. A. (2001) Regulatory functions of 3″ UTRs. Biochem. Biophys. Res. Commun. 288, 291–295.
Bartel, D. P. (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281–297.
Sobell, H. M. (1985) Actinomycin and DNA transcription. Proc. Natl. Acad. Sci. USA 82, 528–5331.
Atasoy, U., Curry, S. L., Lopez de Silanes, I., Shyu, A. B., Casolaro, V., Gorospe, M., and Stellato, C. (2003) Regulation of eotaxin gene expression by TNF-α and IL-4 through mRNA stabilization: involvement of the RNA-binding protein HuR. J. Immunol. 171, 4369–4378.
Grigull, J., Mnaimneh, S., Pootoolal, J., Robinson, M. D., and Hughes, T. R. (2004) Genome-wide analysis of mRNA stability using transcription inhibitors and microarrays reveals posttranscriptional control of ribosome biogenesis factors. Mol. Cell. Biol. 24, 5534–5547.
Nonet, M., Scafe, C., Sexton, J., and Young, R. (1987) Eukaryotic RNA polymerase conditional mutant that rapidly ceases mRNA synthesis. Mol. Cell. Biol. 7, 1602–1611.
Herrick, D., Parker, R., and Jacobson, A. (1990) Identification and comparison of stable and unstable mRNAs in Saccharomyces cerevisiae. Mol. Cell. Biol. 10, 2269–2284.
Herruer, M. S., Mager, W. H., Raue, H. A., Vreken, P., Wilms, E., and Planta, R. J. (1988) Mild temperature shock affects transcription of yeast ribosomal protein genes as well as the stability of their mRNAs. Nucleic Acids Res. 16, 7917–7929.
Adams, C. C. and Gross, D. S. (1991) The yeast heat shock response is induced by conversion of cells to spheroplasts and by potent transcriptional inhibitors. J. Bacteriol. 173, 7429–7435.
Gossen, M. and Bujard, H. (1992) Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc. Natl. Acad. Sci. USA 89, 5547–5551.
Gari, E., Piedrafita, L., Aldea, M., and Herrero, E. (1997) A set of vectors with a tetracycline-regulatable promoter system for modulated gene expression in Saccharomyces cerevisiae. Yeast 13, 837–848.
Loflin, P. T., Chen, C. Y., Xu, N., and Shyu, A. B. (1999) Transcriptional pulsing approaches for analysis of mRNA turnover in mammalian cells. Methods 17, 11–20.
Heaton, B., Decker, C., Muhlrad, D., Donahue, J., Jacobson, A., and Parker, R. (1992) Analysis of chimeric mRNAs identifies multiple regions within the STE3 mRNA which promote rapid mRNA decay. Nucleic Acids Res. 20, 5365–5373.
Decker, C. J. and Parker, R. (1993) A turnover pathway for both stable and unstable mRNAs in yeast: evidence for a requirement for deadenylation. Genes Dev. 7, 1632–1643.
Felici, F., Cesareni, G., and Hughes, J. M. X. (1989) The most abundant small cytoplasmic RNA of Saccharomyces cerevisiae has an important function required for normal cell growth. Mol. Cell. Biol. 9, 3260–3268.
Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A., and Speleman, F. (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3, research 0034.1–0034.11.
Wong, M. L. and Medrano, J. F. (2005) Real-time PCR for mRNA quantitation. BioTechniques 39, 75–85.
Leclerc, G. J., Leclerc, G. M., and Barredo, J. C. (2002) Real-time RT-PCR analysis of mRNA decay: half-life of Beta-actin mRNA in human leukemia CCRF-CEM and Nalm-6 cell lines. Cancer Cell Int. 2, 1.
http://www.gene-quantification.info/
Guthrie, C. and Fink, G., eds. (2002) Guide to Yeast Genetics and Molecular and Cell Biology, Part B. Methods Enzymol. 350.
Amberg, D. C., Burke, D. J., and Strathern, J. N. (2005) Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Acknowledgments
I thank Carol Wilusz for technical assistance with the mammalian RNA and qPCR protocols, and Randi Ulbricht and Florencia Lopez Leban for assistance with manuscript preparation. This work was supported by a grant from the National Institutes of Health (GM63759).
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2008 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Olivas, W.M. (2008). Identification of Changes in Gene Expression by Quantitation of mRNA Levels. In: Wilusz, J. (eds) Post-Transcriptional Gene Regulation. Methods In Molecular Biology™, vol 419. Humana Press. https://doi.org/10.1007/978-1-59745-033-1_17
Download citation
DOI: https://doi.org/10.1007/978-1-59745-033-1_17
Publisher Name: Humana Press
Print ISBN: 978-1-58829-783-9
Online ISBN: 978-1-59745-033-1
eBook Packages: Springer Protocols