The 60S ribosomal protein L13 is the most preferable reference gene to investigate gene expression in selected organs from turkeys and chickens, in context of different infection models

Veterinary Research, Oct 2016

Evaluation of reference genes for expression studies in chickens and turkeys is very much limited and unavailable for various infectious models. In this study, eight candidate reference genes HMBS, HPRT1, TBP, VIM, TFRC, RPLP0, RPL13 and RPS7 were evaluated by five different algorithms (GeNorm, NormFinder, BestKeeper©, delta CT, RefFinder) to assess their stability. In order to analyze a broad variation of tissues, spleen, liver, caecum and caecal tonsil of different aged specific pathogen free (SPF) layer chickens and commercial turkeys, uninfected or infected with the extracellular pathogen Histomonas meleagridis, were included. For tissue samples from SPF chickens RPL13 and TBP were found to be the most stable reference genes. Further testing of RPL13 and TBP in the same organs of uninfected and infected SPF broiler chickens with the intracellular pathogen fowl aviadenovirus confirmed this finding. In tissue samples from turkeys, a stable expression of RPL13 and TFRC genes was noticed. Overall, the determined reference genes should be considered whenever gene expression studies in spleen, liver, caecum and caecal tonsil of chickens and turkeys are performed.

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The 60S ribosomal protein L13 is the most preferable reference gene to investigate gene expression in selected organs from turkeys and chickens, in context of different infection models

Mitra et al. Vet Res The 60S ribosomal protein L13 is the most preferable reference gene to investigate gene expression in selected organs from turkeys and chickens, in context of different infection models Taniya Mitra 0 2 Ivana Bilic 0 2 Michael Hess 0 1 2 Dieter Liebhart 0 2 0 Department for Farm Animals and Veterinary Public Health, Clinic for Poultry and Fish Medicine, University of Veterinary Medicine Vienna , Veterinärplatz 1, 1210, Vienna , Austria 1 Christian Doppler Laboratory for Innovative Poultry Vaccines (IPOV), University of Veterinary Medicine Vienna , Veterinärplatz 1, 1210, Vienna , Austria 2 Department for Farm Animals and Veterinary Public Health, Clinic for Poultry and Fish Medicine, University of Veterinary Medicine Vienna , Veterinärplatz 1, 1210, Vienna , Austria Evaluation of reference genes for expression studies in chickens and turkeys is very much limited and unavailable for various infectious models. In this study, eight candidate reference genes HMBS, HPRT1, TBP, VIM, TFRC, RPLP0, RPL13 and RPS7 were evaluated by five different algorithms (GeNorm, NormFinder, BestKeeper©, delta CT, RefFinder) to assess their stability. In order to analyze a broad variation of tissues, spleen, liver, caecum and caecal tonsil of different aged specific pathogen free (SPF) layer chickens and commercial turkeys, uninfected or infected with the extracellular pathogen Histomonas meleagridis, were included. For tissue samples from SPF chickens RPL13 and TBP were found to be the most stable reference genes. Further testing of RPL13 and TBP in the same organs of uninfected and infected SPF broiler chickens with the intracellular pathogen fowl aviadenovirus confirmed this finding. In tissue samples from turkeys, a stable expression of RPL13 and TFRC genes was noticed. Overall, the determined reference genes should be considered whenever gene expression studies in spleen, liver, caecum and caecal tonsil of chickens and turkeys are performed. - Gene expression analysis provides insights into complex biological regulatory processes and has become an essential part in various molecular biology studies. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is, in many studies, the method of choice for the detection and quantification of mRNA [1]. However, this method can be affected by technical variations in template quantity, quality, reverse transcription process and data analysis which impede correct measurements of true biological deviations [2, 3]. It is therefore essential to normalize these variations. There are several methods to eliminate technically induced variations from the true biological diversity such as in  situ calibration, generic normalization against total mRNA, measuring DNA content of total nucleic acid or normalization with validated reference genes [4]. According to minimum information for publication of quantitative real-time pcr experiments (MIQE) guidelines, a reliable method of normalization uses reference genes which should be validated for every species and also on the basis of different experimental treatments [5]. The use of a single reference gene is considered to be an improper approach for gene expression studies, and the application of several genes for normalization is highly recommended to avoid erroneous results introduced by technical manipulation of samples [4, 6]. In recent years, reference genes were established for different animal species, such as cattle, pig, sheep, goat, horse and fish [7–12]. Also in chickens © 2016 The Author(s). This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. (Gallus gallus), a number of studies validated reference genes under different physiological conditions [13–20]. However, the assessment of genes used for normalization of gene expression during infection with an extracellular pathogen is completely lacking. Furthermore, only a single study evaluated reference genes for brain tissue in turkeys (Meleagris gallopavo) [18]. This prompted us to validate reference genes in spleen, liver, caecum and caecal tonsils from healthy and infected SPF layer chickens and turkeys with the extracellular pathogen Histomonas meleagridis at different ages. In SPF broiler chickens, preselected reference genes were further evaluated using samples from birds infected with the intracellular pathogen fowl aviadenovirus (FAdV). Materials and methods Sample selection A total of 252 different tissue samples f (...truncated)


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Taniya Mitra, Ivana Bilic, Michael Hess, Dieter Liebhart. The 60S ribosomal protein L13 is the most preferable reference gene to investigate gene expression in selected organs from turkeys and chickens, in context of different infection models, Veterinary Research, 2016, pp. 105, 47, DOI: 10.1186/s13567-016-0388-z