**4. Conclusions**

88 Aquaculture

Fig. 3. Ranking of reference genes according to their expression stability in fast muscle of Atlantic cod reared under different photoperiod regimes. The average expression stability

**3.2 Influence of normalisation on photic-induced expression of** *myhc* **in cod muscle**  Myosin is a ubiquitous eukaryotic motor that interacts with actin to generate the force for cellular movements as diverse as cytokinesis and muscle contraction (Cheney et al., 1993). This motor protein accounts for the majority of myofibrils, which themselves make up to two-thirds of muscle protein synthesis (Mommsen, 2001). For this reason, *myhc* has been used to study muscle growth and development in teleosts (Johnston, 2001). Environmental stimuli such as light influence most of the physiological processes in fish and muscle development is not an exception. The influence of photoperiod manipulation on the muscle physiology of Atlantic cod was assessed in this study by profiling the expression of this

For comparison, raw expression data of *myhc* were normalised in two different ways: i) with the use of a two-gene normalisation factor from the most stable genes (*arp* and *ubi*) and, ii) with the least stable reference genes (Fig. 4). Using the best two-gene normalisation factor from geNorm, it was observed that from 0 h to 1 week the expression of *myhc* did not change significantly in either photoperiod regime. However, after a month of photoperiod manipulation, a significant difference was noted between treatments and the group exposed to normal photoperiod showed a significantly higher *myhc* expression than the group

values were calculated with (A) geNorm and (B) NormFinder.

gene during a photoperiod manipulation experiment.

Though morphometric analysis is still an acceptable strategy in studying muscle growth in fish, molecular approaches have opened a new set of possibilities to study this phenomenon and to understand the role of key regulatory molecules in myogenesis. qPCR analysis is the most reliable method to quantify gene expression, provided that suitable reference genes are used for data normalisation. To the best of our knowledge, this report represents the first validation of reference genes for qPCR quantification of muscle transcripts in Atlantic cod reared under different photoperiod regimes.

NormFinder and geNorm identified *ubi* and *arp* as the most suitable gene pair to normalise our expression data. Using this two-gene normalisation factor, a 40% difference in *myhc* transcript levels was observed between photoperiod conditions, which was not detected when data were normalised with *gapdh*. Therefore, it is clear that using inadequate reference genes for normalisation of qPCR data can lead to biologically meaningless conclusions.

This study represents a valuable resource for future gene expression studies aimed at investigating the molecular mechanisms of the photic-plasticity of muscle development in Atlantic cod. Moreover, it is applicable to more general related topics in aquaculture research, including growth and nutrition.

#### **5. Acknowledgments**

This research was funded by the GrowCod Project awarded to Jorge M.O. Fernandes by the Research Council of Norway (ref. 190350). The invaluable help of Dr Alessia Giannetto (University of Messina, Italy) and Dr Lech Kirtiklis (University of Warmia and Mazury in Olsztyn, Poland) is acknowledged. The technical assistance of Marion Nilsen is also acknowledged. The authors would like to thank Bjørnar Eggen, Dalia Dahle and Hilde Ribe at Mørkvedbukta Research Station (Bodø, Norway) for their assistance with the photoperiod experiment.

#### **6. References**


validation of reference genes for qPCR quantification of muscle transcripts in Atlantic cod

NormFinder and geNorm identified *ubi* and *arp* as the most suitable gene pair to normalise our expression data. Using this two-gene normalisation factor, a 40% difference in *myhc* transcript levels was observed between photoperiod conditions, which was not detected when data were normalised with *gapdh*. Therefore, it is clear that using inadequate reference genes for normalisation of qPCR data can lead to biologically meaningless conclusions.

This study represents a valuable resource for future gene expression studies aimed at investigating the molecular mechanisms of the photic-plasticity of muscle development in Atlantic cod. Moreover, it is applicable to more general related topics in aquaculture

This research was funded by the GrowCod Project awarded to Jorge M.O. Fernandes by the Research Council of Norway (ref. 190350). The invaluable help of Dr Alessia Giannetto (University of Messina, Italy) and Dr Lech Kirtiklis (University of Warmia and Mazury in Olsztyn, Poland) is acknowledged. The technical assistance of Marion Nilsen is also acknowledged. The authors would like to thank Bjørnar Eggen, Dalia Dahle and Hilde Ribe at Mørkvedbukta Research Station (Bodø, Norway) for their assistance with the photoperiod

Andersen, C.L., Jensen, J.L. & Orntoft, T.F. (2004). Normalisation of real-time quantitative

Aursnes, I.A., Rishovd, A.L., Karlsen, H.E. & Gjøen, T. (2011). Validation of reference genes

Bustin, S.A. (2002). Quantification of mRNA using real-time reverse transcription PCR (RT-

Bustin, S.A. & Nolan, T. (2004). Pitfalls of quantitative real-time reverse-transcription

Campos, C., Valente, L.M., Borges, P., Bizuayehu, T. & Fernandes, J.M. (2010). Dietary lipid

*l.*) during temperature stress. *BMC Research Notes*, 4, ISSN 1756-0500 Begtashi, I., Rodriguez, L., Moles, G., Zanuy, S. & Carrillo, M. (2004). Long-term exposure to

sets. *Cancer Research*, 64, 15, 5245-5250, ISSN 0008-5472

reverse transcription-PCR data: A model-based variance estimation approach to identify genes suited for normalisation, applied to bladder and colon cancer data

for quantitative RT-qPCR studies of gene expression in Atlantic cod (*Gadus morhua* 

continuous light inhibits precocity in juvenile male European sea bass (*Dicentrarchus labrax, L.*). I. Morphological aspects. *Aquaculture*, 241, 1-4, 539-559,

PCR): trends and problems. *Journal of Molecular Endocrinology*, 29, 1, 23-39, ISSN

polymerase chain reaction. *Journal of Biomolecular Techniques*, 15, 3, 155-166, ISSN

levels have a remarkable impact on the expression of growth-related genes in Senegalese sole (*Solea senegalensis* Kaup). *The Journal of Experimental Biology*, 213, 2,

reared under different photoperiod regimes.

research, including growth and nutrition.

**5. Acknowledgments** 

experiment.

**6. References** 

ISSN 0044-8486

0952-5041

1524-0215

200-209, ISSN 1477-9145

