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Quantitative RT-PCR profiling of the rabbit immune response: assessment of acute Shigella flexneri infection.

Schnupf P, Sansonetti PJ - PLoS ONE (2012)

Bottom Line: Quantitative reverse transcription PCR analysis is an important tool to monitor changes in gene expression in animal models.Recently, increasing coverage of the genome has permitted the prediction of a growing number of genes that are relevant in the context of the immune response.Importantly, all primer pairs were designed to be used under identical experimental conditions, thereby enabling the simultaneous analysis of all genes in a high-throughput format.

View Article: PubMed Central - PubMed

Affiliation: Unité de Pathogénie Microbienne Moléculaire, Institut Pasteur, Paris, France.

ABSTRACT
Quantitative reverse transcription PCR analysis is an important tool to monitor changes in gene expression in animal models. The rabbit is a widely accepted and commonly used animal model in the study of human diseases and infections by viral, fungal, bacterial and protozoan pathogens. Only a limited number of rabbit genes have, however, been analyzed by this method as the rabbit genome sequence remains unfinished. Recently, increasing coverage of the genome has permitted the prediction of a growing number of genes that are relevant in the context of the immune response. We hereby report the design of twenty-four quantitative PCR primer pairs covering common cytokines, chemoattractants, antimicrobials and enzymes for a rapid, sensitive and quantitative analysis of the rabbit immune response. Importantly, all primer pairs were designed to be used under identical experimental conditions, thereby enabling the simultaneous analysis of all genes in a high-throughput format. This tool was used to analyze the rabbit innate immune response to infection with the human gastrointestinal pathogen Shigella flexneri. Beyond the known inflammatory mediators, we identified IL-22, IL-17A and IL-17F as highly upregulated cytokines and as first responders to infection during the innate phase of the host immune response. This set of qPCR primers also provides a convenient tool for monitoring the rabbit immune response during infection with other pathogens and other inflammatory conditions.

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Gene expression changes in rabbit illeal loops infected with avirulent or virulent Shigella.Transcriptional response at A) early (4 to 5.5 hrs) and B) late (8 hrs) time points post infection with the avirulent BS176 strain or the virulent M90T strain. The fold induction in gene expression over uninfected (UI) control loops (Δ-Δ-Ct) are given as the median of twelve samples obtained from six rabbits in three independent experiments. Basal level expressions are listed as the median Ct value above the housekeeping gene GAPDH (Δ-Ct) from twelve uninfected loops at the early time point from six rabbits. As GAPDH is an abundant transcript, smaller changes in fold Ct compared to GAPDH signify more abundant gene transcripts. Increased shading highlights increasing upregulation of statistically significan values while stripes signify downregulation. The statistical significance (p-value) was calculated using the Wilcoxon Signed Rank Test.
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pone-0036446-g001: Gene expression changes in rabbit illeal loops infected with avirulent or virulent Shigella.Transcriptional response at A) early (4 to 5.5 hrs) and B) late (8 hrs) time points post infection with the avirulent BS176 strain or the virulent M90T strain. The fold induction in gene expression over uninfected (UI) control loops (Δ-Δ-Ct) are given as the median of twelve samples obtained from six rabbits in three independent experiments. Basal level expressions are listed as the median Ct value above the housekeeping gene GAPDH (Δ-Ct) from twelve uninfected loops at the early time point from six rabbits. As GAPDH is an abundant transcript, smaller changes in fold Ct compared to GAPDH signify more abundant gene transcripts. Increased shading highlights increasing upregulation of statistically significan values while stripes signify downregulation. The statistical significance (p-value) was calculated using the Wilcoxon Signed Rank Test.

Mentions: We designed and tested twenty-four real-time PCR primer pairs for a quantitative gene expression analysis of key rabbit genes involved in the innate and adaptive immune response to infection (Table 1). We assayed the expression of three chemokines [IL-8, chemokine (C-C motif) ligand (CCL)-4, and CCL20], sixteen cytokines [Interleukin (IL)-1β IL-2, IL-4, IL-6, Il-10, IL-12p35, IL12/23p40, IL-17A, IL-17F, IL-18, IL-21, IL-22, Interferon (IFN)- β, IFN-γ, Transforming growth factor (TGF)-β and Tumor necrosis factor (TNF)-α], three antimicrobials [Leukocyte Protein (LeukoP) p15, neutrophil defensin NP-3α, and the cathelicidin CAP-18] and two enzymes [inducible Nitric oxide synthase (iNOS) and cyclo-oxygenase (COX)-2]. This set of twenty-four genes exhibited a wide range of basal expression levels in uninfected loops (5.9 to 14.8 fold Ct over the expression level of the housekeeping gene GAPDH) (Figure 1). The most abundant transcript at the steady state level was IL-8, followed by CCL4, IL-6, IL-1β and IL-18, while rather low levels were detected for IL-17A, COX-2, IL-4, IFN-β, IL-12/23p40, IL-12p35, IL-2, iNOS and IL-22.


Quantitative RT-PCR profiling of the rabbit immune response: assessment of acute Shigella flexneri infection.

Schnupf P, Sansonetti PJ - PLoS ONE (2012)

Gene expression changes in rabbit illeal loops infected with avirulent or virulent Shigella.Transcriptional response at A) early (4 to 5.5 hrs) and B) late (8 hrs) time points post infection with the avirulent BS176 strain or the virulent M90T strain. The fold induction in gene expression over uninfected (UI) control loops (Δ-Δ-Ct) are given as the median of twelve samples obtained from six rabbits in three independent experiments. Basal level expressions are listed as the median Ct value above the housekeeping gene GAPDH (Δ-Ct) from twelve uninfected loops at the early time point from six rabbits. As GAPDH is an abundant transcript, smaller changes in fold Ct compared to GAPDH signify more abundant gene transcripts. Increased shading highlights increasing upregulation of statistically significan values while stripes signify downregulation. The statistical significance (p-value) was calculated using the Wilcoxon Signed Rank Test.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3366964&req=5

pone-0036446-g001: Gene expression changes in rabbit illeal loops infected with avirulent or virulent Shigella.Transcriptional response at A) early (4 to 5.5 hrs) and B) late (8 hrs) time points post infection with the avirulent BS176 strain or the virulent M90T strain. The fold induction in gene expression over uninfected (UI) control loops (Δ-Δ-Ct) are given as the median of twelve samples obtained from six rabbits in three independent experiments. Basal level expressions are listed as the median Ct value above the housekeeping gene GAPDH (Δ-Ct) from twelve uninfected loops at the early time point from six rabbits. As GAPDH is an abundant transcript, smaller changes in fold Ct compared to GAPDH signify more abundant gene transcripts. Increased shading highlights increasing upregulation of statistically significan values while stripes signify downregulation. The statistical significance (p-value) was calculated using the Wilcoxon Signed Rank Test.
Mentions: We designed and tested twenty-four real-time PCR primer pairs for a quantitative gene expression analysis of key rabbit genes involved in the innate and adaptive immune response to infection (Table 1). We assayed the expression of three chemokines [IL-8, chemokine (C-C motif) ligand (CCL)-4, and CCL20], sixteen cytokines [Interleukin (IL)-1β IL-2, IL-4, IL-6, Il-10, IL-12p35, IL12/23p40, IL-17A, IL-17F, IL-18, IL-21, IL-22, Interferon (IFN)- β, IFN-γ, Transforming growth factor (TGF)-β and Tumor necrosis factor (TNF)-α], three antimicrobials [Leukocyte Protein (LeukoP) p15, neutrophil defensin NP-3α, and the cathelicidin CAP-18] and two enzymes [inducible Nitric oxide synthase (iNOS) and cyclo-oxygenase (COX)-2]. This set of twenty-four genes exhibited a wide range of basal expression levels in uninfected loops (5.9 to 14.8 fold Ct over the expression level of the housekeeping gene GAPDH) (Figure 1). The most abundant transcript at the steady state level was IL-8, followed by CCL4, IL-6, IL-1β and IL-18, while rather low levels were detected for IL-17A, COX-2, IL-4, IFN-β, IL-12/23p40, IL-12p35, IL-2, iNOS and IL-22.

Bottom Line: Quantitative reverse transcription PCR analysis is an important tool to monitor changes in gene expression in animal models.Recently, increasing coverage of the genome has permitted the prediction of a growing number of genes that are relevant in the context of the immune response.Importantly, all primer pairs were designed to be used under identical experimental conditions, thereby enabling the simultaneous analysis of all genes in a high-throughput format.

View Article: PubMed Central - PubMed

Affiliation: Unité de Pathogénie Microbienne Moléculaire, Institut Pasteur, Paris, France.

ABSTRACT
Quantitative reverse transcription PCR analysis is an important tool to monitor changes in gene expression in animal models. The rabbit is a widely accepted and commonly used animal model in the study of human diseases and infections by viral, fungal, bacterial and protozoan pathogens. Only a limited number of rabbit genes have, however, been analyzed by this method as the rabbit genome sequence remains unfinished. Recently, increasing coverage of the genome has permitted the prediction of a growing number of genes that are relevant in the context of the immune response. We hereby report the design of twenty-four quantitative PCR primer pairs covering common cytokines, chemoattractants, antimicrobials and enzymes for a rapid, sensitive and quantitative analysis of the rabbit immune response. Importantly, all primer pairs were designed to be used under identical experimental conditions, thereby enabling the simultaneous analysis of all genes in a high-throughput format. This tool was used to analyze the rabbit innate immune response to infection with the human gastrointestinal pathogen Shigella flexneri. Beyond the known inflammatory mediators, we identified IL-22, IL-17A and IL-17F as highly upregulated cytokines and as first responders to infection during the innate phase of the host immune response. This set of qPCR primers also provides a convenient tool for monitoring the rabbit immune response during infection with other pathogens and other inflammatory conditions.

Show MeSH
Related in: MedlinePlus