The cDNA encoding for G-protein coupled receptor 54 (GPR54) was cloned from the brains of Southern Bluefin Tuna (SBT) and Yellowtail Kingfish (YTK). The SBT GPR54 has an open reading frame of 1134bp encoding a predicted 378 amino acid peptide, containing seven putative transmembrane domains, a 138 bp 5'UTR and 238 3'UTR. The partial YTK GPR54 cDNA contains 729 bp nucleotide sequence encoding 243 amino acid residues.

A RT-qPCR assay was developed for both SBT and YTK GPR54, as confirmed by primer specificity and high R2 values. The reference gene used, Hprt1, displayed consistent Cq values across most of the different sampling points, however some interaction between the reference gene expression and developmental stage was observed. GPR54 expression levels were determined for two cohorts, an immature group and a pubertal group, at two sampling points. RT-qPCR results showed that YTK brain GPR54 expression levels did not show sexual dimorphism and were significantly higher in the reproductively mature fish at a point past their peak spawning time compared with expression in the immature fish group.

The YTK gonad GPR54 expression level was significantly lower in males from the pubertal fish group during peak spawning time. In SBT, GPR54 expression profile in the brain and the gonad did not vary between immature and reproductively advanced fish. Analysis was carried out using both relative gene expression data and raw Cq. Alternative reference genes would need to be examined for the assay to be reliable across tissues and developmental time points.