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      Transcription of reference genes used for quantitative RT-PCR in Atlantic salmon is affected by viral infection

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          Abstract

          Relative quantification using RT-qPCR is a widely used method for transcription profiling. Transcript levels of target genes in fish after experimental infection is often reported without documentation of stably transcribed reference genes. We present results demonstrating that transcription of typically used reference genes in Atlantic salmon is not stable during experimental infection with salmon pancreas disease virus (SPDV). Transcript levels 0 to 6 weeks after challenge revealed statistically significant changes between time-points that corresponded with a peak in viral load 3 weeks after challenge. The results emphasize the need for thorough method validation prior to transcriptional studies during viral infections.

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          Product differentiation by analysis of DNA melting curves during the polymerase chain reaction.

          A microvolume fluorometer integrated with a thermal cycler was used to acquire DNA melting curves during polymerase chain reaction by fluorescence monitoring of the double-stranded DNA specific dye SYBR Green I. Plotting fluorescence as a function of temperature as the thermal cycler heats through the dissociation temperature of the product gives a DNA melting curve. The shape and position of this DNA melting curve are functions of the GC/AT ratio, length, and sequence and can be used to differentiate amplification products separated by less than 2 degrees C in melting temperature. Desired products can be distinguished from undesirable products, in many cases eliminating the need for gel electrophoresis. Analysis of melting curves can extend the dynamic range of initial template quantification when amplification is monitored with double-stranded DNA specific dyes. Complete amplification and analysis of products can be performed in less than 15 min.
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            Gene loss and evolutionary rates following whole-genome duplication in teleost fishes.

            Teleost fishes provide the first unambiguous support for ancient whole-genome duplication in an animal lineage. Studies in yeast or plants have shown that the effects of such duplications can be mediated by a complex pattern of gene retention and changes in evolutionary pressure. To explore such patterns in fishes, we have determined by phylogenetic analysis the evolutionary origin of 675 Tetraodon duplicated genes assigned to chromosomes, using additional data from other species of actinopterygian fishes. The subset of genes, which was retained in double after the genome duplication, is enriched in development, signaling, behavior, and regulation functional categories. The evolutionary rate of duplicate fish genes appears to be determined by 3 forces: 1) fish proteins evolve faster than mammalian orthologs; 2) the genes kept in double after genome duplication represent the subset under strongest purifying selection; and 3) following duplication, there is an asymmetric acceleration of evolutionary rate in one of the paralogs. These results show that similar mechanisms are at work in fishes as in yeast or plants and provide a framework for future investigation of the consequences of duplication in fishes and other animals.
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              Evaluation of potential reference genes in real-time RT-PCR studies of Atlantic salmon

              Background Salmonid fishes are among the most widely studied model fish species but reports on systematic evaluation of reference genes in qRT-PCR studies is lacking. Results The stability of six potential reference genes was examined in eight tissues of Atlantic salmon (Salmo salar), to determine the most suitable genes to be used in quantitative real-time RT-PCR analyses. The relative transcription levels of genes encoding 18S rRNA, S20 ribosomal protein, β-actin, glyceraldehyde-3P-dehydrogenase (GAPDH), and two paralog genes encoding elongation factor 1A (EF1AA and EF1AB) were quantified in gills, liver, head kidney, spleen, thymus, brain, muscle, and posterior intestine in six untreated adult fish, in addition to a group of individuals that went through smoltification. Based on calculations performed with the geNorm VBA applet, which determines the most stable genes from a set of tested genes in a given cDNA sample, the ranking of the examined genes in adult Atlantic salmon was EF1AB>EF1AA>β-actin>18S rRNA>S20>GAPDH. When the same calculations were done on a total of 24 individuals from four stages in the smoltification process (presmolt, smolt, smoltified seawater and desmoltified freshwater), the gene ranking was EF1AB>EF1AA>S20>β-actin>18S rRNA>GAPDH. Conclusion Overall, this work suggests that the EF1AA and EF1AB genes can be useful as reference genes in qRT-PCR examination of gene expression in the Atlantic salmon.
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                Author and article information

                Journal
                Vet Res
                Veterinary Research
                BioMed Central
                0928-4249
                1297-9716
                2011
                18 January 2011
                : 42
                : 1
                : 8
                Affiliations
                [1 ]Section for immunoprophylaxis, National Veterinary Institute, P.O. Box 750 Sentrum, 0106 Oslo, Norway
                [2 ]Department of Basic Sciences and Aquatic Medicine, Norwegian School of Veterinary Science, P.O. Box 8146 Dep, 0033 Oslo, Norway
                [3 ]Norwegian College of Fishery Science, University of Tromsø, 9037 Tromsø, Norway
                [4 ]Department of Food Safety and Infection Biology, Norwegian School of Veterinary Science, P.O. Box 8146 Dep, 0033 Oslo, Norway
                [5 ]Intervet Norbio AS, Thormøhlensgate 55, 5008 Bergen, Norway
                Article
                1297-9716-42-8
                10.1186/1297-9716-42-8
                3031228
                21314970
                28f05c32-96de-44ae-9aac-10c56b5faaa6
                Copyright ©2011 Løvoll et al; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 30 September 2010
                : 18 January 2011
                Categories
                Short Report

                Veterinary medicine
                Veterinary medicine

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