Fluorescent RNA arbitrarily primed polymerase chain reaction. A new differential display approach to detect contaminant-induced alterations of gene expression in wildlife species.
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.
Crump
Doug
D
National Wildlife Research Center, Canadian Wildlife Service, Carleton University, Ottawa, Ontario, Canada.
Chiu
Suzanne
S
Trudeau
Vance L
VL
Kennedy
Sean W
SW
D000818:Animals; D002478:Cells, Cultured; D046091:Charadriiformes; D002645:Chickens; D004372:Ducks; D004586:Electrophoresis; D004785:Environmental Pollutants / Q000633:toxicity; D005456:Fluorescent Dyes / Q000737:chemistry; D015870:Gene Expression / Q000187:drug effects; D020869:Gene Expression Profiling / Q000191:economics / Q000379:methods*; D011075:Polybrominated Biphenyls / Q000633:toxicity; D012313:RNA / Q000737:chemistry / Q000235:genetics*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012235:Rhodamines / Q000737:chemistry
D004785:Environmental Pollutants; D005456:Fluorescent Dyes; D011075:Polybrominated Biphenyls; D012235:Rhodamines; D012313:RNA