Abstract
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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-016-0388-z) contains supplementary material, which is available to authorized users.
Highlights
Gene expression analysis provides insights into complex biological regulatory processes and has become an essential part in various molecular biology studies
The ranking in selected organs of healthy layer chickens was RPL13 (0.745), TBP (0.978) and TFRC (1.192), whereas it was slightly perturbed in tissues from infected chickens, with TBP (0.377) as the most stable followed by RPL13 (0.509) and TFRC (0.719) (Table 3)
In a first step candidate reference genes, namely TBP, HPRT1 and HMBS, were selected in the present work according to their previously described stability in gene expression studies of various avian tissues or cells, such as muscular tissues, liver and leukocytes isolated from spleen, thymus and Bursa of Fabricius of chickens [13, 14, 17]
Summary
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] 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]. 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)
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