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DEVELOPMENT OF A GENE-EXPRESSION ARRAY FOCUSING ON THE HYPOTHALAMUS-PITUATARY-THYROID AXIS IN XENOPUS LAEVIS
Citation:
Korte, J J., J E. Tietge, AND S J. Degitz. DEVELOPMENT OF A GENE-EXPRESSION ARRAY FOCUSING ON THE HYPOTHALAMUS-PITUATARY-THYROID AXIS IN XENOPUS LAEVIS. Presented at Society of Toxicology 42nd Annual Meeting, Salt Lake City, UT, March 9-13, 2003.
Description:
As recommended by the Endocrine Disruptor Screening and Testing Program Advisory Committee (EDSTAC), the USEPA has been developing a screening test capable of detecting effects of Endocrine Disrupting Chemicals (EDCs) on the hypothalamus-pituitary-thyroid (HPT) axis in Xenopus laevis. As part of this, we developed an oligonucleotide array to assess changes in expression of important thyroid-axis genes, allowing comparison of the results with morphometric and biochemical information. The method consists of hybridizing amplified, biotinylated RNA from test organisms with membrane-bound synthetic DNA oligomers of 65 to 70 bases. The biotin label is detected by chemiluminescence and quantified. Currently, there are two different oligos for each of the 70 different genes represented on the array, and each oligo is spotted twice. When tested on samples from stage 53 to 56 tadpoles, the system shows robust responses from housekeeping genes (actin, elongation factor 1-a, ribosomal protein L-8, etc.), as well as strong responses from genes important to the HPT axis. The method has numerous attractive features: 1) very sensitive, surpassing radioactive methods 2) complete genomic information is not available 3) high flexibility, allowing genes to be added as new sequences become available 4) does not require robotics or other highly specialized/expensive equipment 5) potential to detect hundreds of genes at once 6) fast, the results are available a few hours after the hybridzation step. The array bas been run successfully with RNA from whole tadpoles, tail sections, and even on individual brains weighing only a few milligrams. This abstract does not necessarily reflect EPA policy.