cDNA Amplification by SMART-PCR and Suppression Subtractive Hybridization (SSH)-PCR

  • Andrew Hillmann
  • Eimear Dunne
  • Dermot Kenny
Part of the METHODS IN MOLECULAR BIOLOGY™ book series (MIMB, volume 496)

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.

Key Words

Differential expression cDNA amplification SMART-PCR subtractive hybridization suppressive PCR subtracted cDNA 

References

  1. 1.
    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.Google Scholar
  2. 2.
    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.Google Scholar
  3. 3.
    Wetmur, J. G., Davidson, N. (1968) Kinetics of renaturation of DNA. J Mol Biol 31, 349–370.CrossRefPubMedGoogle Scholar
  4. 4.
    Ji, W., et al. (2002) Efficacy of SSH PCR in isolating differentially expressed genes. BMC Genomics 3, 12.CrossRefPubMedGoogle Scholar
  5. 5.
    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.CrossRefPubMedGoogle Scholar
  6. 6.
    Hillmann, A. G., et al. (2006) Comparative RNA expression analyses from small-scale, single-donor platelet samples. J Thromb Haemost 4, 349–356.CrossRefPubMedGoogle Scholar
  7. 7.
    CLONTECH Laboratories, P.A., CA, USA, PCR-Select cDNA Subtraction kit Kit User Manual. 2007.Google Scholar
  8. 8.
    CLONTECH Laboratories, P.A., CA, USA, SMART PCR cDNA SYNTHESIS Kit User Manual. 2007.Google Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Andrew Hillmann
    • 1
  • Eimear Dunne
    • 1
  • Dermot Kenny
    • 2
  1. 1.Regenerative Medicine Institute (REMEDI)National University of IrelandIreland
  2. 2.Molecular and Cellular TherapeuticsRoyal College of Surgeons in IrelandIreland

Personalised recommendations