title: Elasmobranch qPCR reference genes: a case study of hypoxia preconditioned epaulette sharks.
abstract: BACKGROUND
  Elasmobranch fishes are an ancient group of vertebrates which have high potential as model species for research into evolutionary physiology and genomics. However, no comparative studies have established suitable reference genes for quantitative PCR (qPCR) in elasmobranchs for any physiological conditions. Oxygen availability has been a major force shaping the physiological evolution of vertebrates, especially fishes. Here we examined the suitability of 9 reference candidates from various functional categories after a single hypoxic insult or after hypoxia preconditioning in epaulette shark (Hemiscyllium ocellatum).
  
  
  RESULTS
  Epaulette sharks were caught and exposed to hypoxia. Tissues were collected from 10 controls, 10 individuals with single hypoxic insult and 10 individuals with hypoxia preconditioning (8 hypoxic insults, 12 hours apart). We produced sequence information for reference gene candidates and monitored mRNA expression levels in four tissues: cerebellum, heart, gill and eye. The stability of the genes was examined with analysis of variance, geNorm and NormFinder. The best ranking genes in our study were eukaryotic translation elongation factor 1 beta (eef1b), ubiquitin (ubq) and polymerase (RNA) II (DNA directed) polypeptide F (polr2f). The performance of the ribosomal protein L6 (rpl6) was tissue-dependent. Notably, in one tissue the analysis of variance indicated statistically significant differences between treatments for genes that were ranked as the most stable candidates by reference gene software.
  
  
  CONCLUSIONS
  Our results indicate that eef1b and ubq are generally the most suitable reference genes for the conditions and tissues in the present epaulette shark studies. These genes could also be potential reference gene candidates for other physiological studies examining stress in elasmobranchs. The results emphasise the importance of inter-group variation in reference gene evaluation.
authors.affiliation: Division of Genetics and Physiology, Department of Biology, University of Turku, FI-20014 Turku, Finland. katury@utu.fi
authors.forename: Kalle T
authors.initials: KT
authors.lastname: Rytkönen
authors.forename: Gillian M C
authors.initials: GM
authors.lastname: Renshaw
authors.forename: Kevin J
authors.initials: KJ
authors.lastname: Ashton
authors.forename: Grant
authors.initials: G
authors.lastname: Williams-Pritchard
authors.forename: Erica H
authors.initials: EH
authors.lastname: Leder
authors.forename: Mikko
authors.initials: M
authors.lastname: Nikinmaa
mesh_terms: D000818:Animals; D005393:Fish Diseases / Q000235:genetics*; D020869:Gene Expression Profiling; D000860:Hypoxia / Q000235:genetics / Q000662:veterinary*; D020648:Peptide Elongation Factor 1 / Q000235:genetics; D016133:Polymerase Chain Reaction; D012319:RNA Polymerase II / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D012754:Sharks / Q000235:genetics* / Q000502:physiology; D025801:Ubiquitin / Q000235:genetics
chemical_list: D020648:Peptide Elongation Factor 1; D012333:RNA, Messenger; D025801:Ubiquitin; D012319:RNA Polymerase II