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Identification of appropriate reference genes for local immune-related studies in Morada Nova sheep infected with Haemonchus contortus

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Abstract

Due to the great economic impact of Haemonchus contortus on sheep farming, there is an increasing number of studies addressing host resistance against this nematode, including identification of directly related immune mechanisms. In this context, relative gene expression by RT-qPCR have been largely used, due to its rapidity, high sensitivity, specificity, and reproducibility. Although, appropriate reference gene selection is crucial for accurate interpretation of results. In this study, five reference genes (GAPDH, G6PDH, YWHAZ, ACTB, and B2M) were tested for expression stability in abomasum (fundic and pyloric regions) and abomasal lymph nodes of Morada Nova sheep classified as resistant (n = 5) or susceptible (n = 5) to H. contortus infection in a flock of 151 animals. GAPDH combined with YWHAZ were selected as reference genes for abomasal fundic region and abomasal lymph nodes, whereas YWHAZ was the most stable gene for abomasal pyloric region. These genes presented the lowest intra- and inter-group variations and, consequently, highest stability. In contrast, expression of G6PDH was the least stable in all tissues. The impact of reference gene selection was demonstrated by relative quantification of a pro-inflammatory cytokine (TNFα) in abomasal fundic region. Significant differences in TNFα expression levels between resistant and susceptible groups were only observed when the most stable genes (GAPDH combined with YWHAZ) or GAPDH were used as reference genes, whereas no significant differences were observed when other tested reference genes were used. It was demonstrated that normalization of expression data using inappropriate reference genes may significantly influence interpretation results.

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Acknowledgements

The present study was funded by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), through a research grant (Grant No. 2017/01626-1) and scholarships (Grant Nos. 2017/00373-2 and 2017/24289-0), and scholarships from Conselho Nacional de Desenvolvimento Científico e Tecnológico (PIBIC/CNPq, Grant Nos. 122027/2017-5 and 118297/2015-5).

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Authors and Affiliations

  1. Universidade Estadual Paulista Júlio de Mesquita Filho, Via de Acesso Prof. Paulo Donato Castellane, s/n, Jaboticabal, SP, 14884-900, Brazil

    João Henrique Barbosa Toscano

  2. Centro Universitário Central Paulista, Rua Miguel Petroni, 5111, São Carlos, SP, 13563-470, Brazil

    Louyse Gabrielli Lopes, Luciana Aparecida Giraldelo & Matheus Henrique da Silva

  3. Embrapa Pecuária Sudeste, Rodovia Washington Luiz, Km 234 s/n, Fazenda Canchim, PO Box 339, São Carlos, SP, 13560-970, Brazil

    Cintia Hiromi Okino & Ana Carolina de Souza Chagas

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  1. João Henrique Barbosa Toscano
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Correspondence to João Henrique Barbosa Toscano.

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All procedures were approved by the Embrapa Pecuária Sudeste Ethics Committee on Animal Experimentation (process no. 04/2017), in accordance with the ethical principles and guidelines for animal experimentation adopted by the Brazilian College of Experimentation.

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Toscano, J.H.B., Lopes, L.G., Giraldelo, L.A. et al. Identification of appropriate reference genes for local immune-related studies in Morada Nova sheep infected with Haemonchus contortus. Mol Biol Rep 45, 1253–1262 (2018). https://doi.org/10.1007/s11033-018-4281-x

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