A quantitative real-time RT-PCR assay for European eel tyrosine hydroxylase.

• Published in: General and Comparative Endocrinology
• Main Authors: Weltzien, Finn-Arne, Pasqualini, Catherine, Vernier, Philippe, Dufour, Sylvie
• Other Authors: Biologie des organismes marins et écosystèmes (BOME), Muséum national d'Histoire naturelle (MNHN)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS), Développement, évolution et plasticité du système nerveux (DEPSN), Centre National de la Recherche Scientifique (CNRS), Institut de Neurobiologie Alfred Fessard (INAF)
• Format: Article in Journal/Newspaper
• Language: English
• Published: HAL CCSD 2005
• Subjects: Phosphoproteins; Ribosomal Proteins; phosphoprotein P0; Cytochromes b; Algorithms; Amino Acid Sequence; Anguilla metabolism; Animals; Brain anatomy & histology enzymology; Cloning; Molecular; Cytochromes b genetics; DNA Complementary biosynthesis genetics; Female; Molecular Sequence Data; Phosphoproteins genetics metabolism; RNA biosynthesis isolation & purification; Reverse Transcriptase Polymerase Chain Reaction; Ribosomal Proteins genetics metabolism; Tyrosine 3-Monooxygenase; [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]; European eel
• Online Access: https://hal.science/hal-00018371; https://doi.org/10.1016/j.ygcen.2004.12.019

Dopamine (DA) plays a key inhibitory role in pubertal development of the European eel, but how DAergic neuronal activity is regulated is not known in this species. In order to investigate the regulation of DA inhibition at the molecular level, we developed a quantitative real-time RT-PCR (qrtRT-PCR) assay, using the Light Cycler system, for the expression of eel tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis. Two different reference genes were compared: the previously cloned eel cytochrome b, and eel acidic ribosomal phosphoprotein P0, the latter of which we cloned and partly sequenced. To further validate the assay, different methods of total RNA extraction were tested and compared. When applied to cDNA extracted from dissected brains of juvenile eels, the expression of TH was highest in the olfactory bulb, followed by the telencephalon including preoptic area, and the di-/mesencephalic areas excluding the optic lobes. TH expression in the optic lobes and in the medulla oblongata was low, whereas no expression could be detected in corpus cerebellum. This distribution pattern is in agreement with earlier studies on TH in the eel using immunohistochemistry, RT-PCR, and Northern blotting. The developed qrtRT-PCR assay provides a new tool for understanding the mechanisms regulating central DA inhibition of puberty in juvenile eels.