Tu1832 Stability of Reference Genes for Messenger RNA Quantification by Real-Time PCR in Mouse Dextran Sodium Sulfate Experimental Colitis

Eissa, Nour; Hussein, Hayam; Rabbi, Mohammad Fazle; Munyaka, Peris M.; Khafipour, Azin; Bernstein, Çharles N.; Ghia, Jean–Eric

doi:10.1016/s0016-5085(16)33226-7

Cited by 8 publications

(5 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…DNBS is less hazardous than TNBS and can be used safely in a well-ventilated room with personnel wearing protective clothing, gloves and goggles 6 . In our study, we evaluated the suitability of 13 genes, including the most commonly-used reference genes Gapdh, Actb, Rplp0, β2m , and Hprt , which have been used in normalizing mRNA expression in normal and pathological intestinal mucosa 33 34 35 .…”

Section: Discussionmentioning

confidence: 99%

Appropriateness of reference genes for normalizing messenger RNA in mouse 2,4-dinitrobenzene sulfonic acid (DNBS)-induced colitis using quantitative real time PCR

Eissa

Kermarrec

Hussein

et al. 2017

Sci Rep

Self Cite

View full text Add to dashboard Cite

2,4-Dinitrobenzene sulfonic acid (DNBS)-induced colitis is an experimental model that mimics Crohn’s disease. Appropriateness of reference genes is crucial for RT-qPCR. This is the first study to determine the stability of reference gene expression (RGE) in mice treated with DNBS. DNBS experimental Colitis was induced in male C57BL/6 mice. RNA was extracted from colon tissue and comprehensive analysis of 13 RGE was performed according to predefined criteria. Relative colonic TNF-α and IL-1β mRNA levels were calculated. Colitis significantly altered the stability of mucosal RGE. Commonly used glyceraldehyde-3-phosphate dehydrogenase (Gapdh), β-actin (Actb), or β2-microglobulin (β2m) showed the highest fluctuation within the inflamed and control groups. Conversely, ribosomal protein large P0 (Rplp0), non-POU domain containing (Nono), TATA-box-binding protein (Tbp) and eukaryotic translation elongation factor 2 (Eef2) were not affected by inflammation and were the most stable genes. TNF-α and IL-1β mRNA levels was dependent on the reference gene used and varied from significant when the most stable genes were used to non-significant when the least stable genes were used. The appropriate choice of RGE is critical to guarantee satisfactory normalization of RT-qPCR data when using DNBS-Model. We recommend using Rplp0, Nono, Tbp, Hprt and Eef2 instead of common reference genes.

show abstract

Section: Discussionmentioning

confidence: 99%

Appropriateness of reference genes for normalizing messenger RNA in mouse 2,4-dinitrobenzene sulfonic acid (DNBS)-induced colitis using quantitative real time PCR

Eissa

Kermarrec

Hussein

et al. 2017

Sci Rep

Self Cite

View full text Add to dashboard Cite

show abstract

“…A real-time quantitative PCR was used to quantify gene expression in a Roche light cycler 96 Real-Time System using Power SYBR green master mix (Life Technologies, Burlington, ON, Canada). Differences in the threshold cycle (ΔCt) number between the target genes and mouse eukaryotic elongation factor 2 ( Eef2 ) and human TATA-box binding protein ( TBP ) (optimal reference genes) ( 43 – 45 ) were used to normalize expression. Human and mice primers sequences for the genes that encode cytokines, TJ proteins and IECs markers are provided in Tables 1 and 2 .…”

Section: Methodsmentioning

confidence: 99%

Chromofungin Ameliorates the Progression of Colitis by Regulating Alternatively Activated Macrophages

et al. 2017

Self Cite

View full text Add to dashboard Cite

Ulcerative colitis (UC) is characterized by a functional dysregulation of alternatively activated macrophage (AAM) and intestinal epithelial cells (IECs) homeostasis. Chromogranin-A (CHGA) secreted by neuroendocrine cells is implicated in intestinal inflammation and immune dysregulation. CHGA undergoes proteolytic processing to generate CHGA-derived peptides. Chromofungin (CHR: CHGA47–66) is a short CHGA-derived peptide encoded by CHGA Exon-IV and is involved in innate immune regulation, but the basis is poorly investigated. We investigated the expression of CHR in colonic tissue of patients with active UC and assessed the effects of the CHR in dextran sulfate sodium (DSS) colitis in mice and on macrophages and human colonic epithelial cells. We found that mRNA expression of CHR correlated positively with mRNA levels of AAM markers and gene expression of tight junction (TJ) proteins and negatively with mRNA levels of interleukin (IL)-8, IL-18, and collagen in patients with active UC. Moreover, AAM markers correlated positively with gene expression of TJ proteins and negatively with IL-8, IL-18, and collagen gene expression. Experimentally, intracolonic administration of CHR protected against DSS-induced colitis by priming macrophages into AAM, reducing colonic collagen deposition, and maintaining IECs homeostasis. This effect was associated with a significant increase of AAM markers, reduction of colonic IL-18 release and conservation of gene expression of TJ proteins. In vitro, CHR enhanced AAM polarization and increased the production of anti-inflammatory mediators. CHR-treated AAM conditioned medium increased Caco-2 cell migration, viability, proliferation, and mRNA levels of TJ proteins, and decreased oxidative stress-induced apoptosis and proinflammatory cytokines release. Direct CHR treatments had the same effect. In conclusion, CHR treatment reduces the severity of colitis and the inflammatory process via enhancing AAM functions and maintaining IECs homeostasis. CHR is involved in the pathogenesis of inflammation in experimental colitis. These findings provide insight into the mechanisms of colonic inflammation and could lead to new therapeutic strategies for UC.

show abstract

“…The effective dose of 2.5 mg/kg/day, as reflected by previously published data related to peptide use, was administrated via intra-rectal (i.r.) administration [ 20 , 26 ]. Control experimental groups received phosphate buffer saline (1% PBS, i.r.).…”

Section: Methodsmentioning

confidence: 99%

“…A quantitative polymerase chain reaction (RT–qPCR) was used to quantify gene expression in a Roche light cycler 96 Real-Time system using power SYBR green master mix (Life Technologies, Burlington, ON, USA). Difference in the threshold cycle (ΔCt) number between the target genes and mouse eukaryotic elongation factor 2 ( Eef2 ) and human TATA box binding protein (TBP) as optimal reference genes [ 26 , 36 , 37 ] were used to calculate differences in the transcript expression using ΔCt method. Human and mice primers sequences for cytokine, AAM, genes of TJ proteins and IECs markers provided in Table 1 and Table 2 .…”

Section: Methodsmentioning

confidence: 99%

Pancreastatin Reduces Alternatively Activated Macrophages, Disrupts the Epithelial Homeostasis and Aggravates Colonic Inflammation. A Descriptive Analysis

et al. 2021

Self Cite

View full text Add to dashboard Cite

Ulcerative colitis (UC) is characterized by modifying alternatively activated macrophages (AAM) and epithelial homeostasis. Chromogranin-A (CHGA), released by enterochromaffin cells, is elevated in UC and is implicated in inflammation progression. CHGA can be cleaved into several derived peptides, including pancreastatin (PST), which is involved in proinflammatory mechanisms. Previously, we showed that the deletion of Chga decreased the onset and severity of colitis correlated with an increase in AAM and epithelial cells’ functions. Here, we investigated PST activity in colonic biopsies of participants with active UC and investigated PST treatment in dextran sulfate sodium (DSS)-induced colitis using Chga−/− mice, macrophages, and a human colonic epithelial cells line. We found that the colonic protein expression of PST correlated negatively with mRNA expression of AAM markers and tight junction (TJ) proteins and positively with mRNA expression of interleukin (IL)-8, IL18, and collagen in human. In a preclinical setting, intra-rectal administration of PST aggravated DSS-induced colitis by decreasing AAM’s functions, enhancing colonic collagen deposition and disrupting epithelial homeostasis in Chga+/+ and Chga−/− mice. This effect was associated with a significant reduction in AAM markers, increased colonic IL-18 release, and decreased TJ proteins’ gene expression. In vitro, PST reduced Chga+/+ and Chga−/− AAM polarization and decreased anti-inflammatory mediators’ production. Conditioned medium harvested from PST-treated Chga+/+ and Chga−/− AAM reduced Caco-2 cell migration, viability, proliferation, and mRNA levels of TJ proteins and increased oxidative stress-induced apoptosis and proinflammatory cytokines release. In conclusion, PST is a CHGA proinflammatory peptide that enhances the severity of colitis and the inflammatory process via decreasing AAM functions and disrupting epithelial homeostasis.

show abstract

Tu1832 Stability of Reference Genes for Messenger RNA Quantification by Real-Time PCR in Mouse Dextran Sodium Sulfate Experimental Colitis

Cited by 8 publications

References 0 publications

Appropriateness of reference genes for normalizing messenger RNA in mouse 2,4-dinitrobenzene sulfonic acid (DNBS)-induced colitis using quantitative real time PCR

Appropriateness of reference genes for normalizing messenger RNA in mouse 2,4-dinitrobenzene sulfonic acid (DNBS)-induced colitis using quantitative real time PCR

Chromofungin Ameliorates the Progression of Colitis by Regulating Alternatively Activated Macrophages

Pancreastatin Reduces Alternatively Activated Macrophages, Disrupts the Epithelial Homeostasis and Aggravates Colonic Inflammation. A Descriptive Analysis

Contact Info

Product

Resources

About