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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Innis, M. A., Gelfand, D. h., and Snisky, J. J. (eds) (1990) PCR protocols. Academic Press, San Diego, CA.
Sambrook, J., Fritsch, E. F., and Maniatus, T. (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring, NY.
Ausbel, F., Brent, R., Kinston, R. E., et al. (eds) (1995) Short protocols in molecular biology. John Wiley & Sons, Inc., New York.
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.
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.
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.
Thomson, E. and Vincent, R. (2005) Reagent volume and plate bias in real-time polymerase chain reaction. Anal. Biochem. 337, 347–350.
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.
Acknowledgments
This work is supported by National Institutes of Health grants NS-048187, NS-039438, and P20 GM-069985.
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
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
Download citation
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