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Understanding how fish respond to and utilize dietary plant ingredients has therefore been a 71

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Commonly used normalization approaches for quantitative reverse transcription polymerase chain reaction analyses include (a) input nucleic acids standardization (ΔC (q) method), (b) normalizing target gene transcript abundance against a single internal reference gene (ΔΔC (q) method), and (c) geometric averaging of multiple reference gene abundance using the geNorm software. We compared these three approaches to examine expression of a negative muscle growth regulator gene, myostatin-I (mstn-I), in the finfish Lates calcarifer, following 4 weeks of nutritional fasting. Interestingly, these three different approaches led to widely divergent data interpretations. When ΔC (q) and subsequently ΔΔC (q) with α-tub as the reference gene were applied to mstn-I expression data, an ∼threefold upregulation of this gene was observed in fasted compared to fed fish. In contrast, mstn-I appeared unchanged when data was normalized against the geometric average of the two apparently most "stable" reference genes (elongation factor-1 α (ef1-α) and rpl8) selected from a panel comprising seven commonly utilized candidate reference genes (18S, cat-D, ef1-α, rpl8, gapdh, ubq, and α-tub). The geNorm software erroneously suggested that ef1-α, rpl8, and ubq were the most "stable" reference genes, whereas ΔC (q) analysis revealed these genes simply to exhibit similar patterns of regulation in response to fasting. The ΔC (q) approach showed that α-tub was the least variable in its expression level between fasted and fed fish after 4 weeks. The present study also highlights the importance of validating internal references for each time point under investigation when applying ΔΔC (q) and suggests that the more cost-effective ΔC (q) normalization approach, if carefully applied, may in fact produce the most biologically valid results.

BackgroundThe present study aimed to explore the impact of dietary docosahexaenoic acid (DHA) on aspects of the metabolism of Atlantic salmon (Salmo salar). The effects of diets containing increasing levels of DHA (1 g kg−1, 3 g kg−1, 6 g kg−1, 10 g kg−1 and 13 g kg−1) on the liver transcriptome of post-smolt salmon was examined to elucidate patterns of gene expression and responses of specific metabolic pathways. Total RNA was isolated from the liver of individual fish and analyzed using a custom gene expression 44K feature Atlantic salmon oligo-microarray.ResultsThe expression of up to 911 unique annotated genes was significantly affected by dietary DHA inclusion relative to a low DHA reference diet. Analysis of a total of 797 unique genes were found with a significant linear correlation between expression level and dietary DHA. Gene-Set Enrichment Analysis (GSEA) identified a range of pathways that were significantly affected by dietary DHA content.ConclusionsPathways that showed a significant response to dietary DHA level included those for long-chain polyunsaturated fatty acid biosynthesis, fatty acid elongation, steroid biosynthesis, glycan biosynthesis, protein export and protein processing in the endoplasmic reticulum. These findings suggest that in addition to clear roles in influencing lipid metabolic pathways, DHA might also have key functional roles in other pathways distinct from lipid metabolism.