Protocol

DNA and RNA Profiling in Human Blood

Volume 496 of the series METHODS IN MOLECULAR BIOLOGY™ pp 223-243

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

  • Andrew HillmannAffiliated withRegenerative Medicine Institute (REMEDI), National University of Ireland
  • , Eimear DunneAffiliated withRegenerative Medicine Institute (REMEDI), National University of Ireland
  • , Dermot KennyAffiliated withMolecular and Cellular Therapeutics, Royal College of Surgeons in Ireland

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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