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Evaluation of Internal Reference Genes for Quantitative Expression Analysis by Real-Time PCR in Ovine Whole Blood
Experimental Zooprophylactic Institute of Piemonte, Liguria and Valle d’Aosta, 10154 Turin, Italy
Department of Animal Pathology, University of Turin, 10095 Grugliasco, Italy
Azienda Sanitaria Locale TO3, Sanità Animale, 10098 Rivoli, Italy
Azienda Sanitaria Locale AT, Sanità Animale, 14100 Asti, Italy
* Author to whom correspondence should be addressed.
Received: 18 October 2011; Accepted: 1 November 2011 / Published: 9 November 2011
Abstract: The use of reference genes is commonly accepted as the most reliable approach to normalize qRT-PCR and to reduce possible errors in the quantification of gene expression. The most suitable reference genes in sheep have been identified for a restricted range of tissues, but no specific data on whole blood are available. The aim of this study was to identify a set of reference genes for normalizing qRT-PCR from ovine whole blood. We designed 11 PCR assays for commonly employed reference genes belonging to various functional classes and then determined their expression stability in whole blood samples from control and disease-stressed sheep. SDHA and YWHAZ were considered the most suitable internal controls as they were stably expressed regardless of disease status according to both geNorm and NormFinder software; furthermore, geNorm indicated SDHA/HPRT, YWHAZ/GAPDH and SDHA/YWHAZ as the best reference gene combinations in control, disease-stressed and combined sheep groups, respectively. Our study provides a validated panel of optimal control genes which may be useful for the identification of genes differentially expressed by qRT-PCR in a readily accessible tissue, with potential for discovering new physiological and disease markers and as a tool to improve production traits (e.g., by identifying expression Quantitative Trait Loci). An additional outcome of the study is a set of intron-spanning primer sequences suitable for gene expression experiments employing SYBR Green chemistry on other ovine tissues and cells.
Keywords: reference genes; Ovis aries; whole blood; real-time qPCR; normalization
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Peletto, S.; Bertuzzi, S.; Campanella, C.; Modesto, P.; Maniaci, M.G.; Bellino, C.; Ariello, D.; Quasso, A.; Caramelli, M.; Acutis, P.L. Evaluation of Internal Reference Genes for Quantitative Expression Analysis by Real-Time PCR in Ovine Whole Blood. Int. J. Mol. Sci. 2011, 12, 7732-7747.
Peletto S, Bertuzzi S, Campanella C, Modesto P, Maniaci MG, Bellino C, Ariello D, Quasso A, Caramelli M, Acutis PL. Evaluation of Internal Reference Genes for Quantitative Expression Analysis by Real-Time PCR in Ovine Whole Blood. International Journal of Molecular Sciences. 2011; 12(11):7732-7747.
Peletto, Simone; Bertuzzi, Simone; Campanella, Chiara; Modesto, Paola; Maniaci, Maria Grazia; Bellino, Claudio; Ariello, Dario; Quasso, Antonio; Caramelli, Maria; Acutis, Pier Luigi. 2011. "Evaluation of Internal Reference Genes for Quantitative Expression Analysis by Real-Time PCR in Ovine Whole Blood." Int. J. Mol. Sci. 12, no. 11: 7732-7747.