**Validation of Endogenous Reference Genes for qPCR Quantification of Muscle Transcripts in Atlantic Cod Subjected to Different Photoperiod Regimes**

Kazue Nagasawa, Carlo Lazado and Jorge M. O. Fernandes *Faculty of Biosciences and Aquaculture, University of Nordland, Bodø Norway* 

#### **1. Introduction**

Atlantic cod (*Gadus morhua*, L.) is a commercially important species worldwide and overfishing has contributed to a decline of wild stocks below sustainable levels. This has stimulated aquaculture production of this species, which has increased remarkably over the last decade to over 20,000 tonnes in 2008 (FAO). Nevertheless, cod farming still faces several production bottlenecks related to larval quality, nutrition, diseases and precocious sexual maturation. The early onset of sexual maturation at around two years in farmed conditions seriously restricts the profitability of the industry. Sexual maturation and the subsequent spawning result in loss of appetite, reduced feed conversion and increased mortality rate (Karlsen et al., 2006), which leads to an increase in the production time required to reach the desired harvest size.

Photoperiod manipulation, typified by continuous light illumination, has been used to delay sexual maturation to some extent in several aquaculture species, including Atlantic salmon (*Salmo salar*, L.) (Endal et al., 2000), European sea bass (*Dicentrachus labrax*, L.) (Begtashi et al., 2004) and Atlantic cod (Davie et al., 2003; Hansen et al., 2001; Norberg et al., 2004). The application of continuous light from the summer solstice prior to maturation is thought to mask the photoperiod signal that acts as a trigger for gonadal development and spawning (Davie et al., 2003). Taranger et al. (2006) have shown that gonadal maturation of cod kept in sea cages can be delayed by three to five months through application of continuous broadspectrum light. In addition to inhibiting sexual maturation, photoperiod manipulation has a direct effect on somatic growth, particularly during juvenile stages (Davie et al., 2007; Taranger et al., 2006). In fact, short-term application of continuous light was found to induce a 5 to 9% increase in body weight when compared to cod reared under normal photoperiod conditions and significant differences can still be observed at harvesting size, nearly three years later (Imsland et al., 2007). In spite of its obvious relevance for the aquaculture industry, the molecular basis of this growth plasticity induced by light cues is not known. In order to better control the precocious sexual maturation of farmed cod, it is crucial to identify the transcriptional networks related to this phenomenon and to understand how they are influenced by photoperiod.

Muscle is the main tissue supporting fish growth. Teleost myogenesis is a complex phenomenon which involves a number of molecules regulating distinct phases of this process. The development and formation of muscle involves either hypertrophy (expansion of muscle fibre by absorption of myoblast nuclei) or hyperplasia (formation of fibres on the surface of an existing muscle fibre ) (Johnston, 1999). The progression of muscle formation is associated with the sequential expression of key genes from the myogenic regulatory factors (MRFs) family, which include *myoblast differentiation 1 (myoD), myogenic factor 5 (myf-5), myogenin (myoG) and myogenic factor 6/myogenic regulatory factor 4 (myf-6/MRF4)* (Watabe, 1999). Another molecule of significant importance in muscle development is myosin heavy chain (*myhc*), which serves as marker of muscle development in several studies (Johnston, 1999). *Myhc* genes code for a family of ATP-dependent motor proteins that are involved in muscle contraction (Ikeda et al., 2007). *Myhc* activity can be used to monitor fish growth, since most fish have a continuous hyperplasic growth throughout their lifespan and *myhc* is actively involved in muscle protein synthesis (Dhillon et al., 2009).

Quantification of transcript levels by real-time PCR (qPCR) is currently the method of choice, since it is reliable and sensitive enough to quantify even lowly expressed mRNAs in small amount of target tissues (Bustin, 2002). For example, in tiger pufferfish (*Takifugu rubripes*, Temminck & Schlegel) this technique has been used to validate suppression subtractive hybridization results (Fernandes et al., 2005), to examine how temperature affects expression of the growth-related genes *myoG* (Fernandes et al., 2006) and *forkhead box protein K1 (foxk1)* (Fernandes et al., 2007a) during embryonic development, and to examine differential regulation of splice variants of the master transcription factor *myoD1* (Fernandes et al., 2007b). In spite of its enormous potential, relative qPCR quantification has several pitfalls that must be carefully considered (Bustin and Nolan, 2004). In particular, selection of suitable reference genes with even expression in all samples is critical to normalise qPCR data and the use of non-validated reference genes can lead to erroneous conclusions that are biologically meaningless (Fernandes et al., 2008). It is a general consensus that a versatile reference gene stable under various experimental conditions does not exist. Before proceeding to quantifying the expression of a target gene, it is necessary to select the most appropriate reference genes for each species and tissue for a particular experimental setup. A sensible practice involves testing multiple genes for each experiment and using statistical applications to identify the best combination of the two or three most stable genes that will be used to normalise qPCR data (Andersen et al., 2004; Vandesompele et al., 2002).

The aim of the present research paper was to identify suitable reference genes for relative quantification by qPCR of growth- and maturation-related genes that may be affected by photoperiod manipulation. Five commonly used reference genes were evaluated, namely: *βactin* (*actb*), *acidic ribosomal protein* (*arp*), *eukaryotic elongation factor 1α* (*eef1a*), *glyceraldehyde-3 phosphate dehydrogenase* (*gapdh*) and *ubiquitin* (*ubi*). Their transcript levels in the above fast muscle samples were determined by qPCR using SYBR chemistry. GeNorm (Vandesompele et al., 2002) and NormFinder (Andersen et al., 2004) were used to evaluate expression stability of above candidate genes. In addition, to demonstrate the importance of using validated reference genes in qPCR analysis, *myhc* expression was examined. Here, we showed the impact of normalisation strategies (i.e., different individual candidate genes versus the normalisation factor from the two best validated reference genes) on *myhc* expression levels and the necessity of validation to select the most stable reference gene in each experimental plot.
