title: Selection of reference genes for reverse transcription quantitative real-time PCR normalization in black rockfish (Sebastes schlegeli).
abstract: Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a technique widely used for quantification of mRNA transcription. Data normalization is an indispensable process for RT-qPCR and reference genes are most commonly used to normalize RT-qPCR and to reduce possible errors generated in the quantification of genes among several proposed methods. To date, RT-qPCR has been used in terms of gene expression studies in black rockfish (Sebastes schlegeli) but the majority of published RT-qPCR studies still lack proper validation of the reference genes. In the present study, mRNA transcription profiles of eight putative reference genes (18S rRNA, ACTB, GAPDH, TUBA, RPL17, EF1A, HPRT, and B2M) were examined using RT-qPCR in different tissues and larvae developmental stages of black rockfish. Three common statistical algorithms (geNorm, NormFinder, and BestKeeper) were used to assess expression stability and select the most stable genes for gene normalization. Two reference genes, RPL17 and EF1A showed high stability in black rockfish tissue analysis, while GAPDH was the least stable gene. During larvae developmental stages, EF1A, RPL17 and ACTB were identified as the optimal reference genes for data normalization, whereas B2M appeared unsuitable as the reference gene. In summary, our results could provide a useful guideline for reference gene selection and enable more accurate normalization of gene expression data in gene expression studies of black rockfish.
authors.affiliation: College of Marine Life Science, Ocean University of China, Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Qingdao 266003, PR China.
authors.forename: Ma
authors.initials: M
authors.lastname: Liman
authors.forename: Wang
authors.initials: W
authors.lastname: Wenji
authors.forename: Liu
authors.initials: L
authors.lastname: Conghui
authors.forename: Yu
authors.initials: Y
authors.lastname: Haiyang
authors.forename: Wang
authors.initials: W
authors.lastname: Zhigang
authors.forename: Wang
authors.initials: W
authors.lastname: Xubo
authors.forename: Qi
authors.initials: Q
authors.lastname: Jie
authors.forename: Zhang
authors.initials: Z
authors.lastname: Quanqi
mesh_terms: D000465:Algorithms; D000818:Animals; D017931:DNA Primers; D005260:Female; D029941:Fish Proteins / Q000235:genetics; D020869:Gene Expression Profiling / Q000379:methods; D008297:Male; D010473:Perciformes / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012984:Software
chemical_list: D017931:DNA Primers; D029941:Fish Proteins
keywords: Black rockfish; Quantitative real-time PCR; Reference genes; Sebastes schlegeli