A quantitative real-time RT-PCR assay for European eel tyrosine hydroxylase.
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.
Weltzien
Finn-Arne
FA
USM 0401, UMR 5178 CNRS/MNHN/UPMC Biologie des Organismes Marins et Ecosystèmes, Département des Milieux et Peuplements Aquatiques, Muséum National d'Histoire Naturelle, 7 rue Cuvier, 75231 Paris Cedex 05, France.
Pasqualini
Catherine
C
Vernier
Philippe
P
Dufour
Sylvie
S
D000465:Algorithms; D000595:Amino Acid Sequence; D000811:Anguilla / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000033:anatomy & histology / Q000201:enzymology; D003001:Cloning, Molecular; D045303:Cytochromes b / Q000235:genetics; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D005260:Female; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D012313:RNA / Q000096:biosynthesis / Q000302:isolation & purification; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012269:Ribosomal Proteins / Q000235:genetics / Q000378:metabolism; D014446:Tyrosine 3-Monooxygenase / Q000032:analysis* / Q000235:genetics / Q000378:metabolism
D018076:DNA, Complementary; D010750:Phosphoproteins; D012269:Ribosomal Proteins; C054675:ribosomal protein P0; D012313:RNA; D045303:Cytochromes b; D014446:Tyrosine 3-Monooxygenase