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      Evaluation of potential reference genes in real-time RT-PCR studies of Atlantic salmon


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          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.


          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 (EF1A A and EF1A B) 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 EF1A B>EF1A A>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 EF1A B>EF1A A>S20>β-actin>18S rRNA>GAPDH.


          Overall, this work suggests that the EF1A A and EF1A B genes can be useful as reference genes in qRT-PCR examination of gene expression in the Atlantic salmon.

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          Validation of housekeeping genes for normalizing RNA expression in real-time PCR.

          Analysis of RNA expression using techniques like real-time PCR has traditionally used reference or housekeeping genes to control for error between samples. This practice is being questioned as it becomes increasingly clear that some housekeeping genes may vary considerably in certain biological samples. We used real-time reverse transcription PCR (RT-PCR) to assess the levels of 13 housekeeping genes expressed in peripheral blood mononuclear cell culture and whole blood from healthy individuals and those with tuberculosis. Housekeeping genes were selected from conventionally used ones and from genes reported to be invariant in human T cell culture. None of the commonly used housekeeping genes [e.g., glyceraldehyde-phosphate-dehydrogenase (GAPDH)] were found to be suitable as internal references, as they were highly variable (>30-fold maximal variability). Furthermore, genes previously found to be invariant in human T cell culture also showed large variation in RNA expression (>34-fold maximal variability). Genes that were invariant in blood were highly variable in peripheral blood mononuclear cell culture. Our data show that RNA specifying human acidic ribosomal protein was the most suitable housekeeping gene for normalizing mRNA levels in human pulmonary tuberculosis. Validations of housekeeping genes are highly specific for a particular experimental model and are a crucial component in assessing any new model.
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            Housekeeping gene variability in normal and cancerous colorectal, pancreatic, esophageal, gastric and hepatic tissues.

            Careful normalization is essential for the accurate quantitation of mRNA levels in biopsy-sized tissue samples. Commonly, normalization of the target gene with an endogenous standard, mainly housekeeping genes (HKGs), is applied. However, differences in the expression levels of endogenous reference genes have been reported between different tissues and pathological states. Therefore, we were challenged to identify a set of endogenous reference genes whose mRNA expression levels would not change significantly between normal and cancerous tissues. Quantitative real-time PCR (Q-RT-PCR) analysis was applied to evaluate the variability in gene expression among 21 classical housekeeping genes in colorectal, pancreatic, esophageal and gastric cancer as well as in liver metastases in comparison to the corresponding normal tissue. Our results indicated that some housekeeping genes were candidates with relatively stable gene expression in several of the investigated tissues but for most of the HKGs under investigation our data have revealed distinct differences in the extent of variability in gene expression between the different tissues and pathological states. However, for each of the five tissues investigated we found a group of genes that were expressed at a constant level thus representing a panel of candidates that we can recommend as housekeeping genes in the respective tissue types. In summary, our results can be used as guidance for other scientists studying various carcinomas for tissue-specific selection of the optimal housekeeping gene (HKG) to be used in normalizing target gene expression.
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              Messenger RNA turnover in eukaryotes: pathways and enzymes.

              The control of mRNA degradation is an important component of the regulation of gene expression since the steady-state concentration of mRNA is determined both by the rates of synthesis and of decay. Two general pathways of mRNA decay have been described in eukaryotes. Both pathways share the exonucleolytic removal of the poly(A) tail (deadenylation) as the first step. In one pathway, deadenylation is followed by the hydrolysis of the cap and processive degradation of the mRNA body by a 5' exonuclease. In the second pathway, the mRNA body is degraded by a complex of 3' exonucleases before the remaining cap structure is hydrolyzed. This review discusses the proteins involved in the catalysis and control of both decay pathways.

                Author and article information

                BMC Mol Biol
                BMC Molecular Biology
                BioMed Central (London )
                17 November 2005
                : 6
                : 21
                [1 ]National Institute of Nutrition and Seafood Research, Nordnesboder 2, N-5005 Bergen, Norway
                [2 ]Department of Biology, University of Bergen, Thormøhlensgate 55, N-5020 Bergen, Norway
                Copyright © 2005 Olsvik 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.

                : 22 July 2005
                : 17 November 2005
                Research Article

                Molecular biology
                Molecular biology


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