**3. Results and discussion**

84 Aquaculture

qPCR reactions were conducted with the primer sets indicated on table 1. Quantification of transcripts were analysed by qPCR with SYBR Green chemistry (SYBR Green I Master, Roche) on a LightCycler® 480 (Roche) as previously described (Campos et al., 2010). Fiftyfold diluted muscle cDNA were run in duplicate, and minus reverse transcriptase and no template controls were included in the reactions. Thermocycling parameters were as follows: 95°C for 15 min, followed by 45 cycles of 15 s at 94°C, 20 s at 60°C and 20 s at 72°C. Five-point standard curves of a 2-fold dilution series (1:1, 1:2, 1:4, 1:8 and 1:16) were prepared from pooled RNA that was reverse transcribed as above. These dilution curves were used to calculate amplification efficiencies of the PCR reactions (Fernandes et al., 2006). Cycle threshold (Ct) values were determined by the LightCycler® 480 software with a

**Gene GenBank Sequence Size (bp) E (%) Reference**  *actb* AJ555463 Fw: TGACCCTGAAGTACCCCATC 162 77 (Lilleeng et al.,

*arp* EX741373 Fw: TGATCCTCCACGACGATGAG 113 86 (Olsvik et al.,

*eef1a* CO541820 Fw: CACTGAGGTGAAGTCCGTTG 142 84 (Lilleeng et al.,

*gapdh* AY635584 Fw: GGTCGCAACCGCAAGGT 83 88 (Hall et al.,

*ubi* EX735613 Fw: GGCCGCAAAGATGCAGAT 69 87 (Olsvik et al.,

Raw qPCR data were converted to expression level using the above dilution curves. These were then analysed for expression stability using the statistical applications GeNorm

Differences in expression levels of *gapdh*, *actb*, *eef1a*, *arp, ubi* and *myhc* during the photoperiod manipulation experiment were examined by one-way ANOVA with Holm-Sidak post-hoc tests. ANOVA assumptions were checked prior to carrying out the analyses and when the data did not follow the Gaussian distribution or did not meet the equal variance requirements, a Kruskal-Wallis one-way ANOVA on ranks with Dunn's test for post-hoc comparisons was used instead. Statistical analyses were performed with the SigmaStat statistical package (Systat software, London, UK). In all cases, significance was set

Table 1. Primers used in this study. Primer sequences, Genbank accession numbers,

*myhc* AY093703 Fw: CAGAAGCTATAAAAGGTGTCCG

(Vandesompele et al., 2002) and NormFinder (Andersen et al., 2004).

amplicon sizes and PCR efficiencies are indicated.

**2.3.1 Expression stability analyses** 

**2.3 Data analyses** 

**2.3.2 Statistical analyses** 

at *P* < 0.05.

Rv: TCTTCTCCCTGTTGGCTTTG 2007)

Rv: CAGGGCCTTGGCGAAGA 2008)

Rv: GGGGTCGTTCTTGCTGTCT 2007)

Rv: TGACCGTTGAGCATTTCCTTCT 2006)

Rv: CTGGGCTCGACCTCAAGAGT 2008)

2010) Rv: GCAGCCATTCTTCTTATCCTCCTC

86 81 (Koedijk et al.,

**2.2.3 qPCR amplification** 

fluorescence level arbitrarily set to 1.

#### **3.1 Validation of reference genes**

Specificity of qPCR reactions was confirmed by melting curve analysis, which revealed a single dissociation peak for each gene. Global variation on expression profiles of the candidate reference genes can be observed in Fig.1. Mean Ct values of the candidate reference genes were 19.99, 18.36, 18.03, 17.76 for *actb*, *ubi*, *eef1a* and *arp*, respectively, whereas the median Ct value for *gapdh* (25.96) was above the range of values for the other four genes. Expression of *ubi* showed the least variation across samples, in contrast to *gapdh*, *actb* and *eef1a*.

Detailed expression of the individual reference genes during the 6-month photoperiod manipulation experiment is presented in Fig. 2, showing a differential expression trend between sampling points and photoperiod regimes. There were no significant differences (*P* < 0.05) on the overall expression amongst any of the candidate reference genes (*arp*: *P* = 0.997; *eef1*: *P* = 0.735; *ubi*: *P* = 0.124; *gapdh*: *P* = 0.386; *actb*: *P* = 0.554). It is also important to note that the transcript levels of *actb*, *ubi*, *eef1a* and *arp* were almost the same based on the close range of their Ct values and all showed a perceptible increase on their transcript levels at the last 3 sampling points. In contrast, transcript levels of *gapdh* were relatively lower compared with the other reference genes as characterized by having higher Ct values. There was an apparent difference on the expression of *actb* and *gapdh* between treatments.

Fig. 1. Overall expression patterns of candidate reference genes in the muscle of Atlantic cod reared under different photoperiod regimes. Raw cycle threshold (Ct) qPCR data of individual reference genes in all samples (n=96) are represented as box-and-whisker plots. Median values are indicated by a solid line inside the boxes.

Expression stability indices of the candidate reference genes as assessed by geNorm varied with time and photoperiod regime (Table 2). *Arp* and *ubi* were identified as the most stable reference genes. Specifically, *arp* was the most stable in fish group under normal photoperiod (0 h, 6 h, 1 d, 1 w, 1 m and 6 m), while *ubi* was the most stable in the group

Fig. 2. Expression profiles of potential reference genes in fast muscle of cod kept under two photoperiod regimes: continuous illumination (LL) or normal photoperiod (NL). Data are presented as mean ± SD of the raw cycle threshold (Ct) values of A) *actb*, B) *gapdh*, C) *ubi*, D) *eef1a* and E) *arp* as determined by real-time qPCR (n=6).

reared under continuous light (6 h, 1 d, 1 w, 1 m and 2 m). *Actb* and *gapdh* were two of the least stable reference genes, with *gapdh* as the least stable in 3 sampling points (0 h, 6 h and 1 m) regardless of photoperiod regime. Collating all the stability indices of each reference gene, the order of stability from the most to the least was as follows: *arp* > *ubi* > *eef1a* > *actb* > *gapdh*. Pairwise comparisons revealed that *arp* and *ubi* were the best pair for two-gene normalisation with a joint stability value of 0.138 (Fig. 3A).

The validation software, NormFinder identified *arp* (0.084) and *gapdh* (0.259) as the most and least stable reference genes, respectively. The overall ranking reference genes from the most to the least stable was as follows: *arp* > *ubi* > *actb* > *eef1a* > *gapdh* (Fig. 3B). It was also determined that the best pair of candidate reference genes was *arp* and *ubi* with a joint stability value of 0.084. This pairwise result is similar to the result in geNorm that the best pair for two-gene normalisation was *arp* and *ubi*. It was also found that regardless of photoperiod regimes, *gapdh* was the least stable gene at 0 h, 6 h, 1 m and 4 m, corresponding broadly to the results obtained in geNorm.

Fig. 2. Expression profiles of potential reference genes in fast muscle of cod kept under two photoperiod regimes: continuous illumination (LL) or normal photoperiod (NL). Data are presented as mean ± SD of the raw cycle threshold (Ct) values of A) *actb*, B) *gapdh*, C) *ubi*, D)

reared under continuous light (6 h, 1 d, 1 w, 1 m and 2 m). *Actb* and *gapdh* were two of the least stable reference genes, with *gapdh* as the least stable in 3 sampling points (0 h, 6 h and 1 m) regardless of photoperiod regime. Collating all the stability indices of each reference gene, the order of stability from the most to the least was as follows: *arp* > *ubi* > *eef1a* > *actb* > *gapdh*. Pairwise comparisons revealed that *arp* and *ubi* were the best pair for two-gene

The validation software, NormFinder identified *arp* (0.084) and *gapdh* (0.259) as the most and least stable reference genes, respectively. The overall ranking reference genes from the most to the least stable was as follows: *arp* > *ubi* > *actb* > *eef1a* > *gapdh* (Fig. 3B). It was also determined that the best pair of candidate reference genes was *arp* and *ubi* with a joint stability value of 0.084. This pairwise result is similar to the result in geNorm that the best pair for two-gene normalisation was *arp* and *ubi*. It was also found that regardless of photoperiod regimes, *gapdh* was the least stable gene at 0 h, 6 h, 1 m and 4 m, corresponding

*eef1a* and E) *arp* as determined by real-time qPCR (n=6).

normalisation with a joint stability value of 0.138 (Fig. 3A).

broadly to the results obtained in geNorm.


Table 2. Expression stability indices of the five reference genes, as determined by geNorm and NormFinder applications. Relative mRNA level were determined in fast muscle of cod kept under two photoperiod regimes: continuous illumination (LL) and normal photoperiod (NL). The most and the least stable reference genes are shaded in blue and red, respectively.

In order to obtain more robust conclusions it is advisable to do a parallel validation of reference gene stability using different alternative software applications, since there is no each method uses different mathematical models that can lead to different outputs. For example, NormFinder takes all candidate reference genes into account and ranks them with the intragroup and intergroup variation, whereas geNorm sequentially excludes the worst gene ending with two and ranks genes with the degree of similarity of expression. The overall results from NormFinder and geNorm applied to our data revealed that the most stable genes were *arp* and *ubi* and these two is the most suitable pair for two-gene normalisation. In previous qPCR studies in cod, it was also shown that *arp* and *ubi* were the most stable genes and could be used in studying wild populations of cod living in contaminated areas (Olsvik et al., 2008) and ontogeny in cod larvae (Sæle et al., 2009). To optimise the results in selecting the most suitable reference gene, candidate genes should belong to different biological pathways, so as to minimise errors associated with co-regulation. Co-regulation is still possible between *ubi* and *arp*, since they fall on the same biological pathway as important molecules in protein degradation and elongation step of protein synthesis, respectively. However, since these genes were validated by two applications generating similar results, it is fair to consider them as stable in this experimental setup. Both applications identified *gapdh* as the most unstable reference gene. It has been observed that *gapdh* is regulated under varying physiological conditions, which could render this gene inappropriate as reference gene (Olsvik et al., 2005). In a study in cod where candidate reference genes were evaluated during ontogeny with emphasis on the development of gastrointestinal tract, *gapdh* was also rated as one of the least suitable normalisation genes regardless of categorisation and analysis (Sæle et al., 2009). It is also mentioned that in cod, there are two isoforms of *gapdh*, one that is muscle-specific and the other as a brain-specific. Validation of reference genes in cod exposed to thermal stress revealed that *gapdh* was also the least favourable gene for normalisation (Aursnes et al., 2011).

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

#### **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 gene during a photoperiod manipulation experiment.

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

Fig. 4. Expression of *myhc* in the fast muscle of Atlantic cod subjected to different photoperiod regimes. A) Transcript levels of *myhc* gene normalised using *arp* and *ubi*, the best combination for a two-gene normalisation. B) Transcript levels of *myhc* gene normalised by the least stable reference gene, *gapdh* using ∆∆Ct method, Data are shown as mean ± SD of the normalised values (n=6). Asterisk (\*) indicates that a significant difference was detected between photoperiod treatments (*P < 0.001*).

exposed to continuous light. Expression of *myhc* in the natural photoperiod group was approximately 40% higher than the expression in the continuous light group. From 2 to 6 months, *myhc* expression increased equally in both photoperiod regimes.

No significant difference was noted between light regimes throughout whole photoperiod manipulation experiment when *myhc* expression data were normalised using *gapdh*, the least stable reference gene. This stresses the importance of identifying suitable reference genes for a particular biological system, not only to draw robust conclusions but also to identify subtle and important differences in mRNA levels.
