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Validation of Reference Genes for Real-Time Polymerase Chain Reaction Studies in Atlantic Salmon

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Abstract

Optimization of reference genes for real-time polymerase chain reaction (PCR) studies in fish is strongly needed. We systematically tested β-actin (ACTB), 18S rRNA (18S), β2-microglobulin (B2M), elongation factor 1-α (EF1A), RNA polymerase I and II (RPL1/2), and glycerol 6-phosphate dehydrogenase (G6PDH) for stability in salmon immune relevant tissues and kidney cells (ASK) infected with infectious salmon anemia virus (ISAV), plus in tissues from fish fed thia fatty acids. Transcription of all genes was unchanged in infected and thia fatty acid-treated tissues versus normal tissues. Between tissues, 18S and EF1A were most stable, RPL1 and RPL2 were intermediate, and G6PDH and ACTB and B2M were the least stable. However, only 18S had constant expression in infected cells; the rest significantly down-regulated. Implications of this finding were demonstrated when normalizing major histocompatibility complex (MHC) class I expression in ASK. Software predictions supported a proper normalization is obtained combining 18S, EF1A, and RPL1 in vivo, but for in vitro viral infection assays we recommend using 18S.

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Acknowledgments

This work was financed by the European Commission project QLK2–CT–2002–00838 and Norwegian Research Council project NFR 153090/S40. We thank Anne-Lise Rishovd for technical help and Oyvind Kileng for valuable 18S primers.

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Authors and Affiliations

  1. Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo, 0316, Oslo, Norway

    Sven Martin Jorgensen, Ellen Johanne Kleveland & Tor Gjoen

  2. Department of Basic Sciences and Aquatic Medicine, Norwegian School of Veterinary Sciences, 0033, Oslo, Norway

    Unni Grimholt

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  1. Sven Martin Jorgensen
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  2. Ellen Johanne Kleveland
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  3. Unni Grimholt
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  4. Tor Gjoen
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Correspondence to Tor Gjoen.

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Jorgensen, S.M., Kleveland, E.J., Grimholt, U. et al. Validation of Reference Genes for Real-Time Polymerase Chain Reaction Studies in Atlantic Salmon. Mar Biotechnol 8, 398–408 (2006). https://doi.org/10.1007/s10126-005-5164-4

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  • DOI: https://doi.org/10.1007/s10126-005-5164-4

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