Summary
Differential display polymerase chain reaction (PCR) can facilitate the identification of novel molecular end points related to contaminant exposure in a wide range of species. To date, various differential display methodologies have been described in detail. Herein, we describe a modification of the RNA arbitrarily primed PCR (RAP-PCR) method that involves the fluorescent labeling of cDNA transcripts via 5′ rhodamine-labeled 18-mer arbitrary primers. These arbitrary primers typically bind to the coding regions of cDNA, which simplifies the downstream identification of contaminant-responsive genes. The technique has been aptly named fluorescent RNA arbitrarily primed PCR, FRAP-PCR, and has been successfully utilized with several avian species and RNA sources (e.g., cultured cells, tissue). This straightforward, safe, and cost-effective approach represents a useful alternative to the radiometric-based RAP-PCR method.
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Acknowledgments
This work was supported in part by a Pesticide Science Fund grant (Dr. Pierre Mineau, Environment Canada) and by funding from Environment Canada’s Strategic Technology Applications of Genomics for the Environment (STAGE). We acknowledge Stephanie Jones and Magdalena Jagla for critically reading the chapter.
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Crump, D., Chiu, S., Trudeau, V.L., Kennedy, S.W. (2008). Fluorescent RNA Arbitrarily Primed Polymerase Chain Reaction. In: Martin, C.C., Martin, C.C. (eds) Environmental Genomics. Methods in Molecular Biology, vol 410. Humana Press. https://doi.org/10.1007/978-1-59745-548-0_2
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DOI: https://doi.org/10.1007/978-1-59745-548-0_2
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