Development of a quantitative assay to measure expression ofTI Development of a quantitative assay to measure expression of transforming growth factor beta (TGF-beta) in Lost River sucker (Deltistes luxatus) and shortnose sucker (Chasmistes brevirostris) and evaluation of potential pitfalls in use with field-collected samples

摘要

The Nature Conservancy is in the process of restoring the Williamson River Delta in an attempt to recreate important juvenile habitat for the endangered shortnose sucker Chasmistes breyirostris and the endangered Lost River sucker Deltistes luxatus. Measurement of TGF-beta mRNA expression level was one of the indicators chosen to evaluate juvenile sucker health during the restoration process. TGF-beta mRNA expression level has been correlated with disease status in several laboratory studies and TGF-beta mRNA expression level has been used as a species-specific indicator of immune status in field-based fish health assessments. We describe here the identification of TGF-beta and a possible splice variant from shortnose sucker and from Lost River sucker. The performance of a quantitative RT-PCR assay to measure TGF-beta beta mRNA expression level was evaluated in field-collected spleen and kidney tissue samples. The quality of extracted RNA was higher in tissues harvested in September compared to July and higher in tissues harvested at lower temperature compared to higher temperature. In addition, the expression level of both TGF-beta and 18S as assessed by qRT-PCR was higher in samples with higher quality RNA. TGF-beta mRNA expression was lower in kidney than in spleen in both Lost River sucker and shortnose sucker