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Evaluation of reference genes of Mytilus galloprovincialis and Ruditapes philippinarum infected with three bacteria strainsfor gene expression analysis

Published online by Cambridge University Press:  15 January 2015

Rebeca Moreira ,
Patricia Pereiro ,
María M. Costa ,
Antonio Figueras  and
Beatriz Novoa
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Abstract

Quantitative real-time polymerase chain reaction (qPCR) is probably the most used method for gene expression quantification because of its high sensitivity and specificity. Nevertheless, this technology can undergo experimental errors and variations. Normalization of the results using a reference gene is therefore necessary to minimize these variations. As the study of immune genes in bivalve mollusks has increased in the last years, the establishment of adequate and stable reference genes for bivalves is strongly required. We analyzed the behavior of four putative reference genes: ribosomal RNA 18S, actin, elongation factor 1 − α and α-tubulin. The suitability of these four genes as internal control for qPCR was evaluated in mussel (Mytilus galloprovincialis) and clam (Ruditapes philippinarum) hemocytes after bacterial challenge. Four independent approaches (BestKeeper, GeNorm, NormFinder and DeltaCt ) were used to assess the suitable genes for stable expression. For these particular circumstances, the most stable gene in hemocytes was elongation factor 1 − α for mussels and α-tubulin for clams.

Keywords

Shellfish immunityReference (housekeeping) genesqPCRMytilus galloprovincialisRuditapes philippinarumVibrio sppMicrococcus lysodeikticus
Type
Research Article
Information
Aquatic Living Resources , Volume 27 , Issue 3-4 , July 2014 , pp. 147 - 152
Copyright
© EDP Sciences, IFREMER, IRD 2015

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References

Allam, B.,Paillard, C.,Auffret, M., 2000, Alterations in hemolymph and extrapallial fluid parameters in the Manila clam, Ruditapes philippinarum, challenged with the pathogen Vibrio tapetis. J. Invertebr. Pathol. 76, 63–69. Google Scholar
Andersen, C.L.,Jensen, J.L.,Orntoft, T.F., 2004, Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res. 64, 5245–5250. Google Scholar
Araya, M.T.,Siah, A.,Mateo, D.,Markham, F.,McKenna, P.,Johnson, G.,Berthe, F.C.J., 2008, Selection and evaluation of housekeeping genes for haemocytes of soft-shell clams (Mya arenaria) challenged with Vibrio splendidus. J. Invertebr. Pathol. 99, 326–331. Google Scholar
Balseiro, P.,Moreira, R.,Chamorro, R.,Figueras, A.Novoa, B., 2013, Immune responses during the larval stages of Mytilus galloprovincialis: metamorphosis alters immunocompetence, body shape and behavior. Fish Shellfish Immunol. 35, 438–447. Google Scholar
Bustin, S.A.,Benes, V.,Garson, J.A.,Hellemans, J.,Huggett, J.,Kubista, M.,Mueller, R.,Nolan, T.,Pfaffl, M.W.,Shipley, G.L.,Vandesompele, J.,Wittwer, C.T., 2009, The MIQE Guidelines: Minimum information for publication of quantitative real-time PCR Experiments. Clin. Chem. 55, 611–622. Google Scholar
Costa, M.M.,Novoa, B.,Figueras, A., 2008, Influence of β-glucans on the immune responses of carpet shell clam (Ruditapes decussatus) and Mediterranean mussel (Mytilus galloprovincialis). Fish Shellfish Immunol. 24, 498–505. Google Scholar
Costa, M.M.,Prado-Alvarez, M.,Gestal, C.,Li, H.,Roch, P.,Novoa, B.,Figueras, A., 2009, Functional and molecular immune response of Mediterranean mussel (Mytilus galloprovincialis) haemocytes against pathogen-associated molecular patterns and bacteria. Fish Shellfish Immunol. 26, 515–523. Google Scholar
Cubero-Leon, E.,Ciocan, C.M.,Miner, C.,Rotchell, J.M., 2012, Reference gene selection for qPCR in mussel, Mytilus edulis, during gametogenesis and exogenous estrogen exposure. Environ. Sci. Pollut. Res. Int. 19, 2728–2733. Google Scholar
Dheilly, N.M.,Lelong, C.,Huvet, A.,Favrel, P., 2011, Development of a Pacific oyster (Crassostrea gigas) 31,918-feature microarray: identification of reference genes and tissue-enriched expression patterns. BMC Genomics 12, 468. CrossRefGoogle ScholarPubMed
Du, Y.,Zhang, L.,Xu, F.,Huang, B.,Zhang, G.,Li, L., 2013, Validation of housekeeping genes as internal controls for studying gene expression during Pacific oyster (Crassostrea gigas) development by quantitative real-time PCR. Fish Shellfish Immunol. 34, 939–945. Google Scholar
García-Vallejo, J.J., Van Het Hof, B.,Robben, J., Van Wijk, J.A., Van Die, I.,Joziasse, D.H., Van Dijk, W., 2004, Approach for defining endogenous reference genes in gene expression experiments. Anal. Biochem. 329, 293–299. Google Scholar
Gestal, C.,Roch, P.,Renault, T.,Pallavicini, A.,Paillard, C.,Novoa, B.,Oubella, R.,Venier, P.,Figueras, A., 2008, Study of diseases and the immune system of bivalves using molecular biology and genomics. Rev. Fish. Sci. 16, 131–114. Google Scholar
Gómez-León, J.,Villamil, L.,Lemos, M.L.,Novoa, B.,Figueras, A., 2005, Isolation of V. alginolyticus and Vibrio splendidus from aquacultured carpet shell clam (Ruditapes decussatus) larvae associated with mass mortalities. Appl. Environ. Microbiol. 71, 98–104. Google Scholar
Gueguen, Y.,Cadoret, J.P.,Flament, D.,Barreau-Roumiguiere, C.,Girardot, A.L.,Garnier, J.,Hoareau, A.,Bachere, E.,Escoubas, J.M., 2003, Immune gene discovery by expressed sequence tags generated from hemocytes of the bacteria-challenged oyster, Crassostrea gigas. Gene 303, 139–145. Google Scholar
Huggett, J.,Dheda, K.,Bustin, S.,Zumla, A., 2005, Real-time RT-PCR normalisation; strategies and considerations. Genes Immunity 6, 279–284. Google Scholar
Li, H.,Venier, P.,Prado-Alvarez, M.,Gestal, C.,Toubiana, M.,Quartesan, R.,Borghesan, F.,Novoa, B.,Figueras, A.,Roch, P., 2010, Expression of Mytilus immune genes in response to experimental challenges varied according to the site of collection. Fish Shellfish Immunol. 28, 640–648. Google Scholar
Martínez-Fernández, M.,Bernatchez, L., Rolán-Alvarez, E.,Quesada, H., 2010, Insights into the role of differential gene expression on the ecological adaptation of the snail Littorina saxatilis. BMC Evol. Biol. 10, 356. Google Scholar
Martins, E.,Figueras, A.,Novoa, B.,Santos, R.S.,Moreira, R.,Bettencourt, R., 2014, Comparative study of immune responses in the deep-sea hydrothermal vent mussel Bathymodiolus azoricus and the shallow-water mussel Mytilus galloprovincialis challenged with Vibrio bacteria. Fish Shellfish Immunol. 40, 485–499. Google Scholar
Mauriz, O.,Maneiro, V.,Pérez-Parallé, M. L.,Sánchez, J. L.,Pazos, A. J., 2012, Selection of reference genes for quantitative RT-PCR studies on the gonad of the bivalve mollusc Pecten maximus L. Aquaculture 370-371, 158–165. Google Scholar
Mitter, K.,Kotoulas, G.,Magoulas, A.,Mulero, V.,Sepulcre, P.,Figueras, A.,Novoa, B.,Sarrapoulou, E., 2009, Evaluation of candidate reference genes for qPCR during ontogenesis and of immune-relevant tissues of European seabass (Dicentrarchus labrax). Comp. Biochem. Physiol. B 153, 340–347. Google Scholar
Moreira, R.,Balseiro, P.,Romero, A.,Dios, S.,Posada, D.,Novoa, B.,Figueras, A., 2012a, Gene expression analysis of clams Ruditapes philippinarum and Ruditapes decussatus following bacterial infection yields molecular insights into pathogen resistance and immunity. Dev. Comp. Immunol. 36, 140–149. Google Scholar
Moreira, R.,Balseiro, P.,Planas, J.V.,Fuste, B., Beltran, S.,Novoa, B.,Figueras, A., 2012b, Transcriptomics of in vitro immune-stimulated hemocytes from the Manila clam Ruditapes philippinarum using high-throughput sequencing. Plos One 7, e35009. Google Scholar
Moreira, R.,Milan, M.,Balseiro, P.,Romero, A., Babbucci, M.,Figueras, A.,Bargelloni, L.,Novoa, B, 2014, Gene expression profile analysis of Manila clam (Ruditapes philippinarum) hemocytes after a Vibrio alginolyticus challenge using an immune-enriched oligo-microarray. BMC Genomics 15, 267. Google Scholar
Pfaffl, M.W., 2001, A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29, e45. CrossRefGoogle ScholarPubMed
Pfaffl, M.W.,Tichopad, A.,Prgomet, C.,Neuvians, T.P., 2004, Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper-Excel-based tool using pair-wise correlations. Biotechnol. Lett. 26, 509–515. Google Scholar
Romero, A.,Dios, S.,Poisa-Beiro, L.,Costa, M.M.,Posada, D.,Figueras, A.,Novoa, B., 2011, Individual sequence variability and functional activities of fibrinogen-related proteins (FREPs) in the Mediterranean mussel (Mytilus galloprovincialis) suggest ancient and complex immune recognition models in invertebrates. Dev. Comp. Immunol. 35, 334–344. Google Scholar
Rozen, S.,Skaletsky, H.J., 2000, Primer3 on the WWW for general users and for biologist programmers. Meth. Mol. Biol. 132, 365–386. Google Scholar
Siah, A.,Dohoo, C.,McKenna, P.,Delaporte, M.,Berthe, F.C.J., 2008, Selecting a set of housekeeping genes for quantitative real-time PCR in normal and tetraploid haemocytes of soft-shell clams, Mya arenaria. Fish Shellfish Immunol. 25, 202–207. Google Scholar
Silver, N.,Best, S.,Jiang, J.,Thein, S.L., 2006, Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR. BMC Mol. Biol. 7, 33. Google Scholar
Vandesompele J., De Preter K., Pattyn F., Poppe B., Van Roy N., De Paepe A., Speleman F., 2002, Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3, Research0034. Google Scholar
Waki, T.,Shimokawa, J.,Watanabe, S.,Yoshinaga, T.,Ogawa, K., 2012, Experimental challenges of wild Manila clams with Perkinsus species isolated from naturally infected wild Manila clams. J. Invertebr. Pathol. 111, 50–55. Google Scholar
Winnebeck, E.C.,Millar, C.D.,Warman, G.R., 2010, Why does insect RNA look degraded? J. Insect Sci. 10, 159. Google Scholar
Wu, H.,Ji, C.,Wei, L.,Zhao, J.,Lu, H., 2013, Proteomic and metabolomic responses in hepatopancreas of Mytilus galloprovincialis challenged by Micrococcus luteus and Vibrio anguillarum. J. Proteomics 94, 54–67. Google Scholar