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.
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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
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(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)