Abstract
The comparison of two RNA populations that differ from the effects of a single-independent variable, such as a drug treatment or a specific genetic defect, can identify differences in the abundance of specific transcripts that vary in a population-dependent manner. There are a variety of methods for identifying differentially expressed genes, including microarray, SAGE, qRT-PCR, and DDGE. This protocol describes a potentially less sensitive yet relatively easy and cost-effective alternative that does not require prior knowledge of the transcriptomes under investigation and is particularly applicable when minimal levels of starting material, RNA, are available. RNA input can often be a limiting factor when analyzing RNA from, for example, rigorously purified blood cells. This protocol describes the use of SMART-PCR to amplify cDNA from sub-microgram levels of RNA. The amplified cDNA populations under comparison are then subjected to suppression subtractive hybridization (SSH-PCR), a technique that couples subtractive hybridization with suppression PCR to selectively amplify fragments of differentially expressed genes. The final products are cDNA populations enriched for significantly over-represented transcripts in either of the two input RNA preparations. These cDNA populations may then be cloned to make subtracted cDNA libraries and/or used as probes to screen subtracted cDNA, global cDNA, or genomic DNA libraries.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Chenchik, A., Zhu, Y. Y., Diatchenko, L., et al. (1998) Generation and use of high-quality cDNA from small amounts of total RNA by SMART PCR, in Gene Cloning and Analysis by RT-PCR. BioTechniques Books, MA. 305–319.
Chenchik, A., Moqadam, F., Siebert, P. (1996) A new method for full-length cDNA cloning by PCR, in A Laboratory Guide to RNA: Isolation, Analysis, and Synthesis. Wiley-Liss, Inc. 273–321.
Wetmur, J. G., Davidson, N. (1968) Kinetics of renaturation of DNA. J Mol Biol 31, 349–370.
Ji, W., et al. (2002) Efficacy of SSH PCR in isolating differentially expressed genes. BMC Genomics 3, 12.
Diatchenko, L., et al. (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.
Hillmann, A. G., et al. (2006) Comparative RNA expression analyses from small-scale, single-donor platelet samples. J Thromb Haemost 4, 349–356.
CLONTECH Laboratories, P.A., CA, USA, PCR-Select cDNA Subtraction kit Kit User Manual. 2007.
CLONTECH Laboratories, P.A., CA, USA, SMART PCR cDNA SYNTHESIS Kit User Manual. 2007.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Hillmann, A., Dunne, E., Kenny, D. (2009). cDNA Amplification by SMART-PCR and Suppression Subtractive Hybridization (SSH)-PCR. In: Bugert, P. (eds) DNA and RNA Profiling in Human Blood. METHODS IN MOLECULAR BIOLOGY™, vol 496. Humana Press. https://doi.org/10.1007/978-1-59745-553-4_15
Download citation
DOI: https://doi.org/10.1007/978-1-59745-553-4_15
Publisher Name: Humana Press
Print ISBN: 978-1-934115-93-0
Online ISBN: 978-1-59745-553-4
eBook Packages: Springer Protocols