title: Reference gene selection for quantitative real-time RT-PCR normalization in the half-smooth tongue sole (Cynoglossus semilaevis) at different developmental stages, in various tissue types and on exposure to chemicals.
abstract: Quantitative real time RT-PCR has been described as the most sensitive method for the detection of low abundance mRNA. To date, no reference genes have been screened in the half-smooth tongue sole (Cynoglossus semilaevis). The aim of this study was to select the most stable genes for quantitative real-time RT-PCR. Eight housekeeping genes (18S, TUBA, B2M, ACTB, EF1A, GAPDH, RPL17 and UBCE) were tested at different developmental stages, in different tissues, and following exposure to the drug SB-431542. Using geNorm, BestKeeper and NormFinder software, GAPDH/B2M, GAPDH/18S and UBCE/GAPDH were identified as the most suitable genes from samples taken of different developmental stages while 18S/RPL17 were consistently ranked as the best reference genes for different tissue types. Furthermore, TUBA/B2M, TUBA/UBCE and B2M/TUBA were found to be the most suitable genes in samples treated with the drug, SB-431542 by geNorm, BestKeeper and NormFinder respectively. Across both different developmental stages and tissue types, the combination of 18S and GAPDH was the most stable reference gene analyzed by Ref-Finder. To test and verify the screened reference genes, the expression profiles of LEFTY-normalized to the combination of GAPDH/18S and ACTB were presented. These results will be useful for future gene-expression studies in the half-smooth tongue sole.
authors.affiliation: Key Laboratory of Marine Genetics and Breeding, Ocean University of China, Qingdao, Shandong, China.
authors.forename: Conghui
authors.initials: C
authors.lastname: Liu
authors.affiliation: Key Laboratory of Marine Genetics and Breeding, Ocean University of China, Qingdao, Shandong, China.
authors.forename: Nian
authors.initials: N
authors.lastname: Xin
authors.affiliation: Key Laboratory of Marine Genetics and Breeding, Ocean University of China, Qingdao, Shandong, China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhai
authors.affiliation: Key Laboratory of Marine Genetics and Breeding, Ocean University of China, Qingdao, Shandong, China.
authors.forename: Liming
authors.initials: L
authors.lastname: Jiang
authors.affiliation: Laizhou Mingbo Aquatic CO., Ltd., Laizhou, Shandong, China.
authors.forename: Jieming
authors.initials: J
authors.lastname: Zhai
authors.affiliation: Key Laboratory of Marine Genetics and Breeding, Ocean University of China, Qingdao, Shandong, China.
authors.forename: Quanqi
authors.initials: Q
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Marine Genetics and Breeding, Ocean University of China, Qingdao, Shandong, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Qi
mesh_terms: D000818:Animals; D017931:DNA Primers / Q000235:genetics; D029941:Fish Proteins / Q000235:genetics*; D005412:Flatfishes / Q000235:genetics* / Q000254:growth & development; D020869:Gene Expression Profiling / Q000592:standards*; D018507:Gene Expression Regulation, Developmental; D020043:Genes, Essential / Q000235:genetics*; D009928:Organ Specificity; D009930:Organic Chemicals / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012984:Software
chemical_list: D017931:DNA Primers; D029941:Fish Proteins; D009930:Organic Chemicals; D012333:RNA, Messenger