Computational protocol: Thyroid Hormone Upregulates Hypothalamic kiss2 Gene in the Male Nile Tilapia, Oreochromis niloticus

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Protocol publication

[…] The fish were anesthetized by immersing in a 0.01% solution of tricaine methanesulfonate (MS222; Sigma, St. Louis, MO, USA) and killed by decapitation for sample collection. Total RNA from the tilapia brain (n = 1) was prepared using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). One microgram of isolated RNA was used to synthesize the first-strand cDNA using the ReverTra Ace-a first-strand cDNA Synthesis Kit (Toyobo, Osaka, Japan). Partial cDNA fragments were obtained by PCR using degenerate primers or gene-specific primers designed based on the sequences of kiss genes of fugu, grouper, medaka, and mackerel (Table ). Full-length cDNA sequences were obtained by 5′ and 3′ rapid amplification of cDNA ends (RACE) kit (Invitrogen). For all PCR reactions in this study, amplifications were performed with an initial denaturation step at 94°C for 3 min, followed by 40 cycles of 94°C for 15 s, 55-58°C for 15 s, and 72°C for 30 s. The reaction was ended by a further extension of 10 min at 72°C. The amplification products were purified using the E.Z.N.A. Gel Extraction Kit (Omega BioTek, GA, USA) and ligated into the pTZ57R/T vector (Fermentas, MD, USA). Three different individual positive clones were picked to confirm the sequence information using an ABI 3700 sequencer (Applied Biosystems, Foster City, CA, USA). Putative signal peptides and cleavage sites were predicted using SignalP 3.0. Multiple sequence alignments of amino acids were performed with ClustalX (1.81) program. Protein phylogenetic analysis was conducted with MEGA4 using the neighbor-joining method.Chromosomal location of tilapia kiss2 gene was identified and its gene synteny with kiss2 genes in other teleosts (zebrafish, medaka, O. latipes, and puffer fish, Takifugu rubripes) were examined using the Ensemble Genome Browser.The tilapia kiss2 gene promoter sequence was identified in silico using the Ensemble Genome Browser. The 2.0-kb sequence upstream of the untranslated region was considered to be the putative promoter. The putative promoter sequence was analyzed for conserved regulatory elements using online bioinformatic tools (TESS; TFSearch; SignalScan). […]

Pipeline specifications

Software tools SignalP, Clustal W, TFSearch
Applications Phylogenetics, Genome data visualization
Organisms Oreochromis niloticus
Diseases Hypothyroidism
Chemicals Gonadotropin-Releasing Hormone, Methimazole, Steroids, Triiodothyronine