**2.1 Photoperiod experiment and sampling**

### **2.1.1 Fish husbandry**

82 Aquaculture

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

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

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.

actively involved in muscle protein synthesis (Dhillon et al., 2009).

data (Andersen et al., 2004; Vandesompele et al., 2002).

Atlantic cod juveniles, *Gadus morhua* L. with an initial size of 2.7 ± 0.8 g (mean ± standard deviation [SD], n=123) were divided into six tanks (250 m3) with an open flow system at a density of approximately 130 individuals per tank. Sea water was continuously supplied to each tank at 7.4 ± 0.4 °C. A commercial diet (Amber Neptun, Skretting AS, Stavanger, Norway) was provided daily by automatic belt feeders, at 5% (w/w) body weight of the fish and adjusted on a weekly basis.

### **2.1.2 Photoperiod experiment**

Each group of fish in three tanks was either kept under continuous light (LL) or reared under a normal light regime (NL) that corresponded to natural environmental photoperiod conditions in Bodø (67°N), Norway during 6 months from January to July 2010. Day light time was recreated indoors using white light fluorescent tubes (Aura Light International AB, Karlskrona, Sweden) and controlled by a scheduled timer according to local sunrise and sunset times in Bodø. Light intensity on the central surface of each tank was 120 Lux.

#### **2.1.3 Sample collection**

The fish were killed by immersion in seawater containing 0.2 g·L-1 tricaine methanesulfonate (MS222; Sigma, Oslo, Norway). Fast muscle samples were carefully dissected from the trunk area below the second dorsal fin from six fish at the start of the experiment and 0.5, 1, 7, 30, 60, 120 and 180 days thereafter. Tissues were snap-frozen in liquid nitrogen and stored at – 80 °C until RNA extraction.

#### **2.1.4 Ethics statement**

All procedures of fish rearing and tissue sampling were in accordance with the guidelines set by the National Animal Research Authority (Forsøksdyrutvalget, Norway).

#### **2.2 Real-time PCR (qPCR)**

#### **2.2.1 Primer design**

To validate expression stability of reference genes, five candidate genes (*β-actin* (*actb*), *acidic ribosomal protein* (*arp*), *elongation factor 1 alpha* (*eef1a*), *glyceraldehyde-3-phosphate dehydrogenase* (*gapdh*), *ubiquitin* (*ubi*) and *myosin heavy chain* (*myhc*)) were selected and analysed for qPCR validation.

#### **2.2.2 RNA extraction and cDNA synthesis**

Total RNA was extracted from the fast muscle samples above and used to synthesized cDNA as detailed elsewhere (Campos et al., 2010). Two micrograms of total RNA were used for cDNA synthesis by reverse transcription, following treatment with gDNA wipe out buffer (Qiagen, Nydalen, Sweden) to remove genomic DNA contamination.
