Identification and validation of housekeeping genes as internal control for gene expression in an intravenous LPS inflammation model in chickens

Boever, Sandra De; Vangestel, Carl; Backer, Patrick De; Croubels, Siska; Sys, Stanislas U.

doi:10.1016/j.vetimm.2007.12.002

Cited by 139 publications

(66 citation statements)

References 22 publications

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“…The mRNA levels of 18 cytokines, chemokines, or immune-associated proteins (IL-2, -4, -5, -6, -10, -12p35, and -12p40; granulocyte-macrophage colony-stimulating factor [GM-CSF]; NRAMP [natural resistance macrophageassociated protein]; IL-15, -1␤, -8, -17, -18, and -22; tumor necrosis factor alpha [TNF-␣; also called LITAF]; gamma interferon [IFN-␥]; inducible nitric oxide synthase [iNOS]; and gallinacins 1, 2, 4, and 6) were determined. In addition, expression of three housekeeping genes, coding for GAPDH (glyceraldehyde-3-phosphate dehydrogenase), TATA-binding protein (TBP), and ubiquitin (UB), was determined and used for data normalization (6,13). Primers for cytokine genes were either described previously (4,26) or newly designed with Primer3 software (http://frodo.wi.mit.edu/primer3/), and all primer sequences are listed in Table 1.…”

Section: Methodsmentioning

confidence: 99%

Immune Response of Chicken Gut to Natural Colonization by Gut Microflora and to Salmonella enterica Serovar Enteritidis Infection

et al. 2011

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In commercial poultry production, there is a lack of natural flora providers since chickens are hatched in the clean environment of a hatchery. Events occurring soon after hatching are therefore of particular importance, and that is why we were interested in the development of the gut microbial community, the immune response to natural microbial colonization, and the response to Salmonella enterica serovar Enteritidis infection as a function of chicken age. The complexity of chicken gut microbiota gradually increased from day 1 to day 19 of life and consisted of Proteobacteria and Firmicutes. Salmonella enterica is one of the major causes of human food-borne gastroenteritis worldwide, and since poultry are considered to be the most important source of S. enterica for humans, measures of how to limit S. enterica prevalence in poultry are continuously being sought. The already-in-use measures aiming at the reduction of S. enterica prevalence in poultry include strict hygienic standards and vaccination with attenuated or inactivated vaccines. However, although hygienic standards are important for S. enterica control, at the same time, this represents an issue. Chickens for commercial production are hatched in a clean environment, and unlike all other farm animals, chickens will never get into contact with adult birds to become colonized by the healthy microflora of adults. Colonization of mucosal surfaces in newly hatched chickens is therefore a matter of coincidence, and if a bacterial pathogen appears in the environment, the sterile intestinal tract of a newly hatched chicken represents an empty ecological niche enabling such a pathogen essentially unrestricted multiplication followed by prolonged colonization. This is the reason why the use of competitive exclusion (CE) products enabling early rapid colonization of chickens with healthy adult gut microbiota has been successfully tested in poultry (18,20). The positive effect of CE products has been explained by the ability of bacteria present in these products to compete directly with pathogens and also to stimulate maturation of the gut immune system of newly hatched chickens.The interaction between the immune system of the gut and commensal microbiota in chickens starts immediately after hatching and leads to a low level of inflammation characterized by increased interleukin-8 (IL-8) expression (2). This results in the infiltration of heterophils and lymphocytes into the lamina propria or the gut epithelium and normalization of the gut immune system (3, 16, 30). Infiltrating lymphocytes develop further, depending on the gut flora composition, either in terms of a decreasing ratio of ␣␤ to ␥␦ T lymphocytes in the lamina propria or the gut epithelium (15) or in terms of changes in ␣␤ T-cell receptor repertoires (21). In mice, but not in chickens so far, gut microflora has been reported to induce the Th1 and Th17 arms of the immune response, with IL-17 playing an important role in the maturation of the murine gut immune system (9, 12). Interestingly, IL-17 has be...

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Section: Methodsmentioning

confidence: 99%

Immune Response of Chicken Gut to Natural Colonization by Gut Microflora and to Salmonella enterica Serovar Enteritidis Infection

et al. 2011

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“…Recognizing the importance of reference genes in normalisation of qPCR data, various reference genes have been evaluated for their stable expression under specific conditions in various organisms. Many studies have been conducted in the animal and human health (De Boever et al, 2008;Hong et al, 2008) fields that describe the identification of multiple reference genes for normalisation of qPCR data, but similar reports are scarce in plant research (Ransbotyn et al, 2006;Exposito-Rodriguez et al, 2008).…”

Section: Bestkeeper Analysismentioning

confidence: 99%

“…These tools include geNorm, NormFinder and BestKeeper (Vandesompele et al, 2002;Pfaffl et al, 2004;Andersen & Orntoft, 2004), which is freely available on the web and allows researchers to find the best reference gene for their experiments. A great number of studies describing the identification of multiple reference genes for normalisation of qPCR data using these algorithms have been performed on the animal and human health fields (Hong et al, 2008;De Boever et al, 2008), but similar reports are scarce in plant research (Jain et al, 2006;Ransbotyn et al, 2006;Exposito-Rodriguez et al, 2008).…”

Section: Introductionmentioning

confidence: 99%

Real Time PCR: the Use of Reference Genes and Essential Rules Required to Obtain Normalisation Data Reliable to Quantitative Gene Expression

Rocha¹,

Monteiro-Júnior²,

Freire³

et al. 2015

JMBR

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Quantitative Real-time Polymerase Chain Reaction (qPCR) is an important tool for molecular biology and biotechnology research, widely used to determine the expression levels of mRNA. Two main methods to performing qPCR are largely used: The absolute quantification, in which the mRNA levels are determined by using a standard curve and the relative method, which is based on the use of reference genes. Reference genes are widely expressed in cells of animal and plant tissues and their expression pattern are theoretically unchanged within several situations, which makes them an excellent choice to normalize mRNA quantification data in relative qPCR studies. However, several reports are increasingly showing that the use of only one reference gene in relative qPCR studies should be avoided, because in the real world their expression levels can significantly change from tissue to tissue. Several softwares, such as geNorm, BestKeeper and NormFinder, have been developed to perform data normalisation, and these programs may assist in choosing the most stable reference genes. The aim of this review was to describe the current normalisation strategies used in qPCR assay, as well as to establish essential rules to perform reliable mRNA quantification. Finally, this review show some innovations in the advances on qPCR.

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“…The housekeeping gene, such as ACTB, GAPDH and HPRT1 (Godornes et al 2007;Wang et al 2012;Marimoutou et al 2015), is a stably expressed gene that is experimentally verified in given species and tissue under given experimental conditions (Løvdal and Lillo 2009). However, differences in the tissue and differences in experiment condition should have different choice criteria (Radonic et al 2005;Watson et al 2007;Boever et al 2008;Cinar et al 2012;Bages et al 2015). There is a few reports about the stability of housekeeping genes in chickens (Bages et al 2015;Nascimento et al 2015).…”

Section: Introductionmentioning

confidence: 99%

Selection of housekeeping genes for quantitative gene expression analysis in yellow-feathered broilers

Zhang

Gao

Huang

et al. 2017

Italian Journal of Animal Science

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The aim of this study was designed to select housekeeping genes for quantitative gene expression analysis in yellow-feathered broilers. Twelve 3-week-old chickens were randomly selected from 60 yellow-feathered broilers. Then, 12 chickens were killed; the liver and jejunum samples were collected. The gene expression of housekeeping genes (b-actin, ACTB; glyceraldehyde-3-phosphate dehydrogenase, GAPDH; hypoxanthine phosphoribosyl transferase 1, HPRT1; ribosomal protein L13, RPL13; TATA box binding protein, TBP; hydroxymethylbilane synthase, HMBS) were determined using quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR). Furthermore, the expression stabilities of housekeeping genes were analysed using geNorm, Normfinder and BestKeeper programs. The result showed that RPL13 is the most proper gene in liver, GADPH is the most proper gene in jejunum, and HMBS is the most proper gene in all tissues. In conclusion, this result provides the integrated reported evaluation of housekeeping genes for use in expression studies in yellow-feathered broilers. These findings further emphasise the need to accurately validate candidate housekeeping genes in the study before use in gene expression studies using RT-PCR. ARTICLE HISTORY

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Identification and validation of housekeeping genes as internal control for gene expression in an intravenous LPS inflammation model in chickens

Cited by 139 publications

References 22 publications

Immune Response of Chicken Gut to Natural Colonization by Gut Microflora and to Salmonella enterica Serovar Enteritidis Infection

Immune Response of Chicken Gut to Natural Colonization by Gut Microflora and to Salmonella enterica Serovar Enteritidis Infection

Real Time PCR: the Use of Reference Genes and Essential Rules Required to Obtain Normalisation Data Reliable to Quantitative Gene Expression

Selection of housekeeping genes for quantitative gene expression analysis in yellow-feathered broilers

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