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AU-Differential Display 235
15. To reduce artifacts, it is important to use some hot start techniques or a preblocked
polymerase. Some brands offer this kind of product. Those products that require extra
PCR cycles for activation have been avoided. Note that different polymerases can also
give different band patterns and sensitivity.
16. Although even when fixed and washed radioactive gels still tend to give background
signals on the autorad that assists the orientation, it may be preferred to use luminescent
labels (such as the Glogos labels from Stratagene) for this purpose.
17. According to the scientist’s interests, genes that are downregulated in experimental samples
might also be of major interest. They would be isolated from the control sample since its
concentration there would be higher.
Acknowledgments
Pepi Caro is warmly thanked for her expert technical support and patience during
the development of this work.
This work has been supported in part by the CDTI as Concerted Projects 95/0184
and 97/0313.
References
1. Xu, N., Chen, C. Y., and Shyu, A. B. (1997) Modulation of the fate of cytoplasmic mRNA
by AU-rich elements: key sequence features controlling mRNA deadenylation and decay.
Mol. Cell Biol. 17, 4611– 4621.
2. Shaw, G. and Kamen, R. (1986) A conserved AU sequence from the 3′ untranslated region
of GM-CSF mRNA mediates selective mRNA degradation. Cell 46, 659–667.
3. Caput, D., Beutler, B., Hartog, K., Thayer, R., Brown-Shimer, S., and Cerami, A.
(1986) Identification of a common nucleotide sequence in the 3’-untranslated region of
mRNA molecules specifying inflammatory mediators. Proc. Natl. Acad. Sci. USA 83,
1670–1674.
4. Balmer, L. A., Beveridge, D. J., Jazayeri, J. A., Thomson, A. M., Walker, C. E., and
Leedman, P. J. (2001) Identification of a novel AU-Rich element in the 3′ untranslated
region of epidermal growth factor receptor mRNA that is the target for regulated RNAbinding
proteins. Mol. Cell Biol. 21, 2070–2084.
5. King, L. M. and Francomano, C. A. (2001) Characterization of a human gene encoding
nucleosomal binding protein nsbp1. Genomics 71, 163–173.
6. Liang, P. and Pardee, A. B. (1992) Differential display of eukaryotic messenger RNA by
means of the polymerase chain reaction. Science 257, 967–971.
7. Welsh, J., Chada, K., Dalal, S. S., Cheng, R., Ralph, D., and McClelland, M. (1992)
Arbitrarily primed PCR fingerprinting of RNA. Nucleic Acids Res. 20, 4965– 4970.
8. Stone, B. and Wharton, W. (1994) Targeted RNA fingerprinting: the cloning of differentiallyexpressed
cDNA fragments enriched for members of the zinc finger gene family. Nucleic
Acids Res. 22, 2612–2618.
9. Donohue, P. J., Alberts, G. F., Guo, Y., and Winkles, J. A. (1995) Identification by targeted
differential display of an immediate early gene encoding a putative Serine/Threonine
kinase. J. Biol. Chem. 270, 10,351–10,357.
10. Fischer, A., Saedler, H., and Theissen, G. (1995) Restriction fragment length polymorphismcoupled
domain directed differential display: a highly efficient technique for expression
analysis of multigene families. Proc. Natl. Acad. Sci. USA 92, 5331–5335.