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Differential Gene Expression in ES-Derived Neural Stem Cells by Using RT-PCR

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Book cover Neural Stem Cells

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 438))

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Summary

Embryonic stem (ES) cells hold promise to treat a variety of disease. The major obstacle is to determine the requirements that will drive these cells to a particular lineage. Two approaches to examine lineage commitment are the addition of growth factors or directed differentiation of ES cells. Although many neural genes have been identified, the cascade of gene expression that directs neural differentiation is not well understood. Today, with microarray technology, large data sets of differential gene expression patterns are used to identify genes that may be used as indicators of a particular cell lineage or tissue type. Semiquantitative polymerase chain reaction (PCR) can be carried out to verify the expression of individual genes, followed by quantitative PCR to precisely determine the level of mRNA expression. However, functional analysis of potential neurogenic genes must be done to identify those genes that play a critical role in neural lineage commitment.

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References

  1. Innis, M. A., Gelfand, D. h., and Snisky, J. J. (eds) (1990) PCR protocols. Academic Press, San Diego, CA.

    Google Scholar 

  2. Sambrook, J., Fritsch, E. F., and Maniatus, T. (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring, NY.

    Google Scholar 

  3. Ausbel, F., Brent, R., Kinston, R. E., et al. (eds) (1995) Short protocols in molecular biology. John Wiley & Sons, Inc., New York.

    Google Scholar 

  4. Morrison, T. B., Weis, J. J., and Wittwer, C. T. (1998) Quantitation of low-copy transcripts by continuous SYBR Green I monitoring during amplification. Biotechniques 24, 945–948, 960, 962.

    Google Scholar 

  5. Gratsch, T. E. and O’Shea, K. S. (2002) Noggin and chordin have distinct activities in promoting lineage commitment of mouse embryonic stem (ES) cells. Dev. Biol. 245, 83–94.

    Article  CAS  PubMed  Google Scholar 

  6. Velkey J. M., Slawny, N. S., Gratsch, T. E., and O’Shea, K. S. (2006) Gene silencing using RNA interference in embryonic stem cells. In: Turksen, K. (ed) Methods in molecular biology, vol 329. Humana Press Inc., Totowa, NJ, pp 233–262.

    Google Scholar 

  7. Thomson, E. and Vincent, R. (2005) Reagent volume and plate bias in real-time polymerase chain reaction. Anal. Biochem. 337, 347–350.

    Article  CAS  PubMed  Google Scholar 

  8. Livak, K. J. and Schmittgen, T. D. (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔ CT method. Methods 25, 402–408.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This work is supported by National Institutes of Health grants NS-048187, NS-039438, and P20 GM-069985.

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© 2008 Humana Press, a part of Springer Science+Business Media, LLC

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Slawny, N., Pacut, C., Gratsch, T.E. (2008). Differential Gene Expression in ES-Derived Neural Stem Cells by Using RT-PCR. In: Weiner, L.P. (eds) Neural Stem Cells. Methods in Molecular Biology™, vol 438. Humana Press. https://doi.org/10.1007/978-1-59745-133-8_21

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  • DOI: https://doi.org/10.1007/978-1-59745-133-8_21

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-846-1

  • Online ISBN: 978-1-59745-133-8

  • eBook Packages: Springer Protocols

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