Optimization of differential display polymerase chain reaction as a bioindicator for the cladoceran Daphnia magna.
Research on toxicant-responsive genes is providing new and important bioindicators for environmental biologists. Identifying genes whose expression is modulated by toxicant exposure provides important clues into the mechanisms underlying toxicity. In addition, toxicant-responsive genes can be developed as molecular end points that are likely to be sensitive tools for environmental assessment. Differential display polymerase chain reaction (ddPCR) is a useful approach for screening and analyzing the expression of genes. A ddPCR protocol was optimized to investigate gene expression in the cladoceran Daphnia magna. The modified protocol requires submicrogram quantities of total RNA (from <10 animals) and utilizes a sensitive fluorescent tagging system. By reverse-transcribing total RNA with arbitrary 18-nucleotide primers and PCR-amplifying the cDNA using the same arbitrary primers under low-stringency conditions, reproducible and consistent ddPCR profiles were generated. Minimal variability was introduced by reaction differences or biological variability. A trial stress (starvation) was found to generate modest differences in the ddPCR profiles. This technique promises to significantly advance knowledge regarding gene expression during toxicant insult. Furthermore, this represents the first step in the development of a novel gene fingerprinting technique that can be applied to any compound and organism of interest.
Diener
Lara C
LC
Department of Biology, University of Waterloo, 200 University Avenue West, Waterloo, Ontario, Canada.
Schulte
Patricia M
PM
Dixon
D George
DG
Greenberg
Bruce M
BM
D000818:Animals; D035061:Control Groups; D016172:DNA Fingerprinting; D017931:DNA Primers; D018076:DNA, Complementary / Q000032:analysis; D003621:Daphnia / Q000187:drug effects* / Q000235:genetics; D003851:Deoxyribonucleases; D015870:Gene Expression / Q000187:drug effects; D016133:Polymerase Chain Reaction / Q000379:methods*; D012313:RNA / Q000302:isolation & purification; D012260:Ribonucleases; D014158:Transcription, Genetic; D014873:Water Pollutants / Q000633:toxicity
D017931:DNA Primers; D018076:DNA, Complementary; D014873:Water Pollutants; D012313:RNA; D003851:Deoxyribonucleases; D012260:Ribonucleases