Abstract
Comparing two RNA populations that differ from the effects of a single independent variable, such as a drug treatment or a specific genetic defect, can establish differences in the abundance of specific transcripts that vary in a population dependent manner. There are different methods for identifying differentially expressed genes. These methods include microarray, Serial Analysis of Gene Expression (SAGE), and quantitative Reverse-Transcriptase Polymerase Chain Reaction (qRT-PCR). Herein, the protocol describes an easy and cost-effective alternative that does not require prior knowledge of the transcriptomes under examination. It is specifically relevant when low levels of RNA starting material are available. This protocol describes the use of Switching Mechanism At RNA Termini Polymerase Chain Reaction (SMART-PCR) to amplify cDNA from small amounts of RNA. The amplified cDNA populations under comparison are then subjected to Suppression Subtractive Hybridization (SSH-PCR). SSH-PCR is a technique that couples subtractive hybridization with suppression PCR to selectively amplify fragments of differentially expressed genes. The resulting products are cDNA populations enriched for significantly overrepresented transcripts in either of the two input RNAs. These cDNA populations can then be cloned to generate subtracted cDNA library. Microarrays made with clones from the subtracted forward and reverse cDNA libraries are then screened for differentially expressed genes using targets generated from tester and driver total RNAs.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Diatchenko, L., Lau, Y. F., Campbell, A. P., Chenchik, A., Moqadam, F., Huang, B., Lukyanov, S., Lukyanov, K., Gurskaya, N., Sverdlov, E. D., and Siebert, P. D. (1996) Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries, Proc. Natl Acad. Sci. USA 93, 6025–6030.
Chee, M., Yang, R., Hubbell, E., Berno, A., Huang, X. C., Stern, D., Winkler, J., Lockhart, D. J., Morris, M. S., and Fodor, S. P. (1996) Accessing genetic information with high-density DNA arrays, Science 274, 610–614.
Bautz, E. K., and Reilly, E. (1966) Gene-specific messenger RNA: isolation by the deletion method, Science 151, 328–330.
Duguid, J. R., and Dinauer, M. C. (1990) Library subtraction of in vitro cDNA libraries to identify differentially expressed genes in scrapie infection, Nucl. Ac. Res. 18, 2789–2792.
Ghorbel, M. T., Sharman, G., Hindmarch, C., Becker, K. G., Barrett, T., and Murphy, D. (2006) Microarray screening of suppression subtractive hybridization-PCR cDNA libraries identifies novel RNAs regulated by dehydration in the rat supraoptic nucleus, Physiol. Genomics 24, 163–172.
Acknowledgments
MTG is funded by the Bristol NIHR BRU in Cardiovacular Medicine and the Garfield Weston Foundation.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Ghorbel, M.T., Murphy, D. (2011). Suppression Subtractive Hybridization. In: Merighi, A. (eds) Neuropeptides. Methods in Molecular Biology, vol 789. Humana Press. https://doi.org/10.1007/978-1-61779-310-3_15
Download citation
DOI: https://doi.org/10.1007/978-1-61779-310-3_15
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-309-7
Online ISBN: 978-1-61779-310-3
eBook Packages: Springer Protocols