Elasmobranch qPCR reference genes: A case study of hypoxia preconditioned epaulette sharks

Kalle T. Rytkönen, Gillian M C Renshaw, Kevin J. Ashton, Grant Williams-Pritchard, Erica H. Leder, Mikko Nikinmaa

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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.

Original languageEnglish
Article number27
JournalBMC Molecular Biology
Volume11
DOIs
Publication statusPublished - 23 Apr 2010

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Elasmobranchii
Sharks
Polymerase Chain Reaction
Genes
Peptide Elongation Factor 1
Ubiquitin
Vertebrates
Analysis of Variance
Hypoxia
RNA Polymerase II
Genomics
Cerebellum
Fishes
Software

Cite this

Rytkönen, K. T., Renshaw, G. M. C., Ashton, K. J., Williams-Pritchard, G., Leder, E. H., & Nikinmaa, M. (2010). Elasmobranch qPCR reference genes: A case study of hypoxia preconditioned epaulette sharks. BMC Molecular Biology, 11, [27]. https://doi.org/10.1186/1471-2199-11-27
Rytkönen, Kalle T. ; Renshaw, Gillian M C ; Ashton, Kevin J. ; Williams-Pritchard, Grant ; Leder, Erica H. ; Nikinmaa, Mikko. / Elasmobranch qPCR reference genes : A case study of hypoxia preconditioned epaulette sharks. In: BMC Molecular Biology. 2010 ; Vol. 11.
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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.",
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Elasmobranch qPCR reference genes : A case study of hypoxia preconditioned epaulette sharks. / Rytkönen, Kalle T.; Renshaw, Gillian M C; Ashton, Kevin J.; Williams-Pritchard, Grant; Leder, Erica H.; Nikinmaa, Mikko.

In: BMC Molecular Biology, Vol. 11, 27, 23.04.2010.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Elasmobranch qPCR reference genes

T2 - A case study of hypoxia preconditioned epaulette sharks

AU - Rytkönen, Kalle T.

AU - Renshaw, Gillian M C

AU - Ashton, Kevin J.

AU - Williams-Pritchard, Grant

AU - Leder, Erica H.

AU - Nikinmaa, Mikko

PY - 2010/4/23

Y1 - 2010/4/23

N2 - 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.

AB - 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.

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U2 - 10.1186/1471-2199-11-27

DO - 10.1186/1471-2199-11-27

M3 - Article

VL - 11

JO - BMC Molecular Biology

JF - BMC Molecular Biology

SN - 1471-2199

M1 - 27

ER -