Validation of reference genes for qPCR studies on Caco-2 cell differentiation

Piana, Claudia; Wirth, Michael; Gerbes, Stefan; Viernstein, Helmut; Gabor, Franz; Toegel, Stefan

doi:10.1016/j.ejpb.2008.03.008

Cited by 29 publications

(24 citation statements)

References 9 publications

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“…Nevertheless, recent reports indicated that they were demodulated in different diseases, animal models, or even under varied experimental conditions, which may lead to skewed results and consequently misinterpretations. Genes expressed at stable levels within the cells in the studied experimental system need to be identified and used as reference [37][38][39][40]. To ensure stable RNA transcription in different conditions, we accessed 12 candidates for reference gene under our different conditions and chose YWAHZ for HS181 hESC and GADPH for SHEF1 using NormFinder software.…”

Section: Discussionmentioning

confidence: 99%

Endoplasmic Reticulum Stress Signals in Defined Human Embryonic Stem Cell Lines and Culture Conditions

Blanco-Gelaz

Suárez-Álvarez

Ligero

et al. 2010

Stem Cell Rev and Rep

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Human embryonic stem cells (hESCs) are especially resistant to several cellular stresses, but the existence and induction of Endoplasmic Reticulum (ER) stress by culture conditions are unknown. Using qPCR, here, we investigated the behavior of the principal sensors of ER stress and their relation with the feeder layer, the type of conditioned media used in feeder free systems and the upregulation of several differentiation markers. We observed the preservation of pluripotency, and detected differential expression of differentiation markers in HS181 and SHEF1 hESCs growing on Adipose-derived mesenchymal stem cells (ASCs) and feeder-free system with different conditioned media (HEF-CM and ASC-CM). Taken together, these results demonstrate evidence of ER stress events that cells must resolve to survive and maintenance of markers of pluripotency. The early differentiation status defined could progress into a more differentiated state, and may be influenced by culture conditions.

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Section: Discussionmentioning

confidence: 99%

Endoplasmic Reticulum Stress Signals in Defined Human Embryonic Stem Cell Lines and Culture Conditions

Blanco-Gelaz

Suárez-Álvarez

Ligero

et al. 2010

Stem Cell Rev and Rep

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“…Quantitative RT‐PCR (qRT‐PCR) was performed using the Universal Probe Library (UPL) method with a StepOnePlus RT‐PCR system (Hoffmann La Roche). β‐Actin, a stable housekeeping gene in Caco‐2, was used to normalize the amounts of RNA in each sample (Piana and others ). Fold changes in mRNA levels relative to the control samples were reported.…”

Section: Methodsmentioning

confidence: 99%

Ginger Extract Suppresses Inflammatory Response and Maintains Barrier Function in Human Colonic Epithelial Caco‐2 Cells Exposed to Inflammatory Mediators

Kim

2017

Journal of Food Science

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The beneficial effects of ginger in the management of gastrointestinal disturbances have been reported. In this study, the anti-inflammatory potential of ginger extract was assessed in a cellular model of gut inflammation. In addition, the effects of ginger extract and its major active compounds on intestinal barrier function were evaluated. The response of Caco-2 cells following exposure to a mixture of inflammatory mediators [interleukin [IL]-1β, 25 ng/mL; lipopolysaccharides [LPS], 10 ng/mL; tumor necrosis factor [TNF]-α, 50 ng/mL; and interferon [INF]-γ, 50 ng/mL] were assessed by measuring the levels of secreted IL-6 and IL-8. In addition, the mRNA levels of cyclooxygenase-2 and inducible nitric oxide synthase were measured. Moreover, the degree of nuclear factor (NF)-κB inhibition was examined, and the intestinal barrier function was determined by measuring the transepithelial electrical resistance (TEER) and fluorescein isothiocyanate (FITC)-dextran transfer. It was observed that ginger extract and its constituents improved inflammatory responses by decreasing the levels of nitrite, PGE2, IL-6, and IL-8 via NF-κB inhibition. The ginger extract also increased the TEER and decreased the transfer of FITC-dextran from the apical side of the epithelium to the basolateral side. Taken together, these results show that ginger extract may be developed as a functional food for the maintenance of gastrointestinal health.

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“…An increasing body of literature, however, has reported that some traditional HKGs demonstrated significant species variability in expression levels (Brinkhof et al, 2006;Langnaese et al, 2008;Lee et al, 2002;Piana et al, 2008;Spinsanti et al, 2006). Even within the same species, the expression levels of the traditional HKGs can vary by cell and tissue type as well as experimental condition or time point (Bonefeld et al, 2008;Radonic et al, 2004).…”

mentioning

confidence: 95%

Using microarray technology to select housekeeping genes in Chinese hamster ovary cells

Bahr

Borgschulte

Kayser

et al. 2009

Biotech & Bioengineering

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In the present study, we have identified species-specific housekeeping genes (HKGs) for Chinese Hamster Ovary (CHO) cells using data from microarray gene expression profiling. HKGs suitable for quantitative RT-PCR normalization should display relatively stable expression levels across experimental conditions. We analyzed transcription profiles of several IgG-producing recombinant CHO cell lines under numerous culture conditions using a custom CHO DNA microarray platform and calculated relative expression variability from 124 microarrays. We selected a novel panel of candidate HKGs based on their low variability in expression from the microarray data. Compared to three traditional HKGs (Gapdh, Actb, and B2m), the majority of genes on this panel demonstrated lower or equal variability. Each candidate HKG was then validated using qRT-PCR. Final selection of CHO-specific HKGs include Actr5, Eif3i, Hirip3, Pabpn1, Vezt, Cog1, and Yaf2. The results reported here provide a useful tool for gene expression analyses in CHO cells, a critical expression platform used in biotherapeutics.

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Validation of reference genes for qPCR studies on Caco-2 cell differentiation

Cited by 29 publications

References 9 publications

Endoplasmic Reticulum Stress Signals in Defined Human Embryonic Stem Cell Lines and Culture Conditions

Endoplasmic Reticulum Stress Signals in Defined Human Embryonic Stem Cell Lines and Culture Conditions

Ginger Extract Suppresses Inflammatory Response and Maintains Barrier Function in Human Colonic Epithelial Caco‐2 Cells Exposed to Inflammatory Mediators

Using microarray technology to select housekeeping genes in Chinese hamster ovary cells

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