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RNA pp 123-135 | Cite as

Fractionation of mRNA Based on the Length of the Poly(A) Tail

  • Hedda A. Meijer
  • Cornelia H. de Moor
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 703)

Abstract

Poly(A) tail length plays an important role in mRNA stability and translational control. Poly(A) fractionation is a very powerful technique to separate mRNAs according to the length of the poly(A) tail. Poly(A) fractionation can be used to detect small changes in poly(A) tail length or to prepare samples for microarray analysis. RNA or crude lysate is mixed with biotinylated oligo(dT), which is then bound to paramagnetic streptavidin beads. Oligoadenylated mRNA is eluted first with a high salt buffer, followed by a low salt elution for polyadenylated mRNA. Elution of the RNA in two fractions can be used as a preparation of samples for microarray analysis while elution of the mRNA in several fractions can be used to analyse (changes in) poly(A) tail length. This method allows for accurate quantification of the amount of oligoadenylated/polyadenylated RNA in each fraction because it is not dependent on visualising the smears representing the variations in poly(A) tail length. The method is technically easy, fast, highly reproducible and can be performed on almost any sample containing RNA.

Key words

Poly(A) tail length fractionation translation mRNA stability polysome profiling 

References

  1. 1.
    Piccioni, F., Zappavigna, V., Verrotti, A. C. (2005) Translational regulation during oogenesis and early development: the cap-poly(A) tail relationship. C.R. Biol 328, 863–881.CrossRefPubMedGoogle Scholar
  2. 2.
    Meyer, S., Temme, C. and Wahle, E. (2004) Messenger RNA turnover in eukaryotes: pathways and enzymes. Crit Rev Biochem Mol Biolv 39, 197–216.CrossRefGoogle Scholar
  3. 3.
    Meijer, H.A., Bushell, M., Hill, K., Gant, T.W., Willis, A.E., Jones, P. and De Moor, C.H. (2007) A novel method for poly(A) fractionation reveals a large population of mRNAs with a short poly(A) tail in mammalian cells. Nucleic Acids Res 35, e132.CrossRefPubMedGoogle Scholar
  4. 4.
    Gorgoni, B., Gray, N.K. (2004) The roles of cytoplasmic poly(A)-binding proteins in regulating gene expression: a developmental perspective. Brief Funct Genomics Proteomics 3, 125–141.CrossRefGoogle Scholar
  5. 5.
    Zangar, R.C., Hernandez, M., Kocarek, T.A. and Novak, R.F. (1995) Determination of the poly(A) tail lengths of a single mRNA species in total hepatic RNA. Biotechniques 18, 465–469.PubMedGoogle Scholar
  6. 6.
    Couttet, P., Fromont-Racine, M., Steel, D., Pictet, R. and Grange, T. (1997) Messenger RNA deadenylation precedes decapping in mammalian cells. Proc Natl Acad Sci USA 94, 5628–5633.CrossRefPubMedGoogle Scholar
  7. 7.
    Sallés, F.J., Richards, W.G. and Strickland, S. (1999) Assaying the polyadenylation state of mRNAs. Methods 17, 38–45.CrossRefPubMedGoogle Scholar
  8. 8.
    Rassa, J.C., Wilson, G.M., Brewer, G.A. and Parks, G.D. (2000) Spacing constraints on reinitiation of paramyxovirus transcription: the gene end U tract acts as a spacer to separate gene end from gene start sites. Virology 274, 438–449.CrossRefPubMedGoogle Scholar
  9. 9.
    Piqué, M., López, M. and Méndez, R. (2006) Cytoplasmic mRNA polyadenylation and translation assays. Methods Mol Biol 322, 183–198.CrossRefPubMedGoogle Scholar
  10. 10.
    Belloc, E., Méndez, R. (2008) A deadenylation negative feedback mechanism governs meiotic metaphase arrest. Nature 452, 1017–1022.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Toxicology Unit, Medical Research CouncilHodgkin Building, University of LeicesterLeicesterUK
  2. 2.RNA Biology GroupSchool of Pharmacy, Centre for Biomolecular Sciences, University of NottinghamNottinghamUK

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