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() Transcriptome profiles as a function of xenobiotic exposure in the grass shrimp, Palaemonetes pugio, using Serial Analysis of Gene Expression (SAGE).
Griffitt, R1, Roberts, M2, Xin, G2, Chandler, G1, Quattro, J2, 1 University of South Carolina, Dept. of Environmental Health Sciences, Columbia, SC, USA2 University of South Carolina, Dept. of Biological Sciences
ABSTRACT- We are utilizing the emerging genomics technique Serial Analysis of Gene Expression (SAGE) to investigate the effect of fipronil, a phenylpyrazol insecticide commonly applied to golf courses in the SE Unites States, on the transcriptional state of the grass shrimp, Palaemonetes pugio. Based on the principal that individual mRNAs are uniquely identifiable by short (10-20 bp) sequences, SAGE generates gene expression profile libraries by directly counting the number of short oligonucleotides (tags) that are representative of each of the different mRNAs. The final copy number is a direct, unbiased estimate of each mRNA's proportion of the total mRNA population in the individual, and therefore of the transcriptional state of the parent gene. Direct comparisons between exposed and control SAGE libraries identify differences in mRNA expression profiles indicative of xenobiotic exposure. We exposed 30 male P. pugio (10 males per replicate, 3 replicates per treatment) to a sex-specific fipronil LC50 concentration (2.0 ug/L) for 96 hours, plus acetone controls, with complete water replacement every 24 hours. Observed mortality was 50% in the fipronil exposure, while no control mortality was observed. At the end of the exposure, surviving individuals from both control and exposure treatments were sacrificed live for RNA extraction. RNA quality and concentration were measured, and equal masses of RNA from each of three individuals were pooled for SAGE library construction using the Invitrogen Long SAGE kit. The control and fipronil libraries were constructed simultaneously. We have successfully completed both control and fipronil SAGE libraries, with an estimate of greater than 80,000 tags in each, and are in the process of sequencing the libraries. Direct comparisons of tag frequency between libraries using the Audic-Claverie test will identify genes with altered expression levels following fipronil exposure.
Key words: Palaemonetes pugio, SAGE, toxicogenomics, fipronil
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