title: Quantitative real time RT-PCR study of pathogen-induced gene expression in rock bream (Oplegnathus fasciatus): internal controls for data normalization.
abstract: Rock bream Oplegnathus fasciatus is an important economic fish species. In this study, we evaluated the appropriateness of six housekeeping genes as internal controls for quantitative real-time PCR (RT-qPCR) analysis of gene expression in rock bream before and after pathogen infection. The expression of the selected genes in eight tissues infected with Vibrio alginolyticus or megalocytivirus was determined by RT-qPCR, and the PCR data were analyzed with geNorm and NormFinder algorithms. The results showed that before pathogen infection, mediator of RNA polymerase II transcription subunit 8 and β-actin were ranked as the most stable genes across the examined tissues. After bacterial or viral infection, the stabilities of the housekeeping genes varied to significant extents in tissue-dependent manners, and no single pair of genes was identified as suitable references for all tissues for either of the pathogen stimuli. In addition, for the majority of tissues, the most stable genes during bacterial infection differed from those during viral infection. Nevertheless, optimum reference genes were identified for each tissue under different conditions. Taken together, these results indicate that tissue type and the nature of the infectious agent used in the study can all influence the choice of normalization factors, and that the optimum reference genes identified in this study will provide a useful guidance for the selection of internal controls in future RT-PCR study of gene expression in rock bream.
authors.affiliation: Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, PR China; Graduate University of the Chinese Academy of Sciences, Beijing 100049, PR China.
authors.forename: Bao-cun
authors.initials: BC
authors.lastname: Zhang
authors.affiliation: Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, PR China.
authors.forename: Li
authors.initials: L
authors.lastname: Sun
authors.affiliation: Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, PR China.
authors.forename: Zhi-zhong
authors.initials: ZZ
authors.lastname: Xiao
authors.affiliation: Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, PR China. Electronic address: huyonghua@qdio.ac.cn.
authors.forename: Yong-hua
authors.initials: YH
authors.lastname: Hu
mesh_terms: D000199:Actins / Q000235:genetics / Q000378:metabolism; D000704:Analysis of Variance; D000818:Animals; D017931:DNA Primers / Q000235:genetics; D004266:DNA Virus Infections / Q000235:genetics / Q000378:metabolism / Q000662:veterinary*; D005393:Fish Diseases / Q000235:genetics* / Q000382:microbiology* / Q000821:virology*; D005786:Gene Expression Regulation / Q000235:genetics; D020043:Genes, Essential / Q000235:genetics; D007497:Iridoviridae; D010473:Perciformes; D012319:RNA Polymerase II / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012015:Reference Standards; D014735:Vibrio Infections / Q000235:genetics / Q000378:metabolism / Q000662:veterinary*; D044149:Vibrio alginolyticus
chemical_list: D000199:Actins; D017931:DNA Primers; D012319:RNA Polymerase II
keywords: Housekeeping gene; Internal reference; Oplegnathus fasciatus; Quantitative real time RT-PCR