Use of a quantitative competitive RT-PCR to evaluate aromatase expression in a turtle

MURDOCK, C.*; WIBBELS, T.: Use of a quantitative competitive RT-PCR to evaluate aromatase expression in a turtle

The red eared slider turtle, Trachemys scripta, possess temperature-dependent sex determination (TSD). Previous studies with T. scripta have suggested that steroids, in particular estrogens, may play key roles in the sex determination cascade. Therefore, steroidogenic enzymes involved in the synthesis of estrogens are of particular interest. Previously in our laboratory the cDNA for one such steroidogenic enzyme, aromatase, has been cloned and sequenced from T. scripta. Using this sequence data, a PCR-based strategy was employed in the construction of an RNA competitor molecule for aromatase. The synthesis of this RNA competitor facilitated the development of a quantitative competitive RT-PCR (QC RT-PCR) for aromatase. In the current study, we have validated and begun utilizing this QC RT-PCR. Preliminary results have shown that: (1) When competitor RNA was added to total RNA extracted from tissues, both co-amplified. (2) Varying the amount of competitor while leaving the amount of total RNA added to each tube constant results in varying intensity in the competitor bands, while the intensity of the endogenous target band remains constant. (3) By varying the amount of competitor added to each sample tube, a standard curve of intensity can be generated, by which aromatase expression during TSD can be quantified. We are now using this QC RT-PCR to examine the expression of aromatase during TSD. QC RT-PCRs are currently being performed on total RNA isolated from adrenal-kidney-gonadal complexes taken from embryos incubated at both male and female-producing temperatures (26OC and 31OC respectively) at stages 15, 17, 19, and 21 of embryonic development. The results will provide a chronology of aromatase expression during TSD.

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