Springer Protocols: Abstract: Identification of Changes in Gene Expression by Quantitation of mRNA Levels

Identification of Changes in Gene Expression by Quantitation of mRNA Levels

By: Wendy M. Olivas³

Abstract

Full Text

Download PDF (443K)

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.

Affiliation(s): (3) Department of Biology, University of Missouri-St. Louis, St. Louis, MO, USA

Book Title: Post-Transcriptional Gene Regulation

Series: Methods in Molecular Biology | Volume: 419 | Pub. Date: Dec-21-2007 | Page Range: 243-258 | DOI: 10.1007/978-1-59745-033-1_17

Subject: Genetics/Genomics

Key Words: Steady-state RNA levels - RNA quantitation - mRNA degradation - mRNA decay - northern blot - real-time PCR - qPCR - transcriptional shut off

References

Download References

1.	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.

2.	Grzybowska, E. A., Wilczynska, A., and Siedlecki, J. A. (2001) Regulatory functions of 3″ UTRs. Biochem. Biophys. Res. Commun. 288, 291–295.

3.	Bartel, D. P. (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281–297.

4.	Sobell, H. M. (1985) Actinomycin and DNA transcription. Proc. Natl. Acad. Sci. USA 82, 528–5331.

5.	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.

6.	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.

7.	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.

8.	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.

9.	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.

10.	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.

11.	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.

12.	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.

13.	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.

14.	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.

15.	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.

16.	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.

17.	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.

18.	Wong, M. L. and Medrano, J. F. (2005) Real-time PCR for mRNA quantitation. BioTechniques 39, 75–85.

19.	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.

20.	http://www.gene-quantification.info/

21.	Guthrie, C. and Fink, G., eds. (2002) Guide to Yeast Genetics and Molecular and Cell Biology, Part B. Methods Enzymol. 350.

22.	Amberg, D. C., Burke, D. J., and Strathern, J. N. (2005) Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

Comments (Loading...)

Post comment/View all comments