The striped sea bream (Lithognathus mormyrus) has been recently introduced as a bioindicator fish species in Mediterranean coastal habitats. The purpose of this study was to apply a real-time PCR assay for the determination of absolute levels of hepatic VTG transcript in this fish, identifying minimal and maximal levels and establishing the relationship between VTG RNA levels and ovarian development. A partial VTG cDNA was cloned from hepatic RNA of a striped sea bream female. Specific primers were designed based on its sequence and used for PCR and also for in vitro synthesis of partial VTG RNA standard. Hepatic VTG transcript levels were quantified by real-time PCR, using serially diluted VTG RNA standards for construction of a calibration curve and equation. VTG RNA levels were normalized to total RNA or 18S ribosomal RNA (determined by real-time PCR). VTG RNA was hardly detected in the liver of males, or females with small oocytes (diameter <100 μm). A linear correlation was found between these two parameters at larger oocyte diameter (>150 μm). VTG level reached a maximum of 204 fmol/pmol 18S RNA or 49 fmol/μg RNA. The results demonstrate the wide dynamic range of the established real-time PCR assay.
Bibliographical noteFunding Information:
This study was funded by the European commission Grant EVK3-2001-00057. We are grateful to Dr. N. Denslow, University of Florida, Gainseville, for sharing with us her protocol and degenerate primer sequences for amplification of VTG cDNAs from fish.