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. 2016 May 31;11(5):e0156289.
doi: 10.1371/journal.pone.0156289. eCollection 2016.

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

Nour Eissa  1   2 , Hayam Hussein  3 , Hongxing Wang  1   4 , Mohammad F Rabbi  1 , Charles N Bernstein  5   6 , Jean-Eric Ghia  1   2   5   6
Affiliations

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

Nour Eissa et al. PLoS One. .

Abstract

Background: Many animal models have been developed to characterize the complexity of colonic inflammation. In dextran sodium sulfate (DSS) experimental colitis in mice the choice of reference genes is critical for accurate quantification of target genes using quantitative real time PCR (RT-qPCR). No studies have addressed the performance of reference genes in mice DSS-experimental colitis. This study aimed to determine the stability of reference genes expression (RGE) in DSS-experimental murine colitis.

Methods: Colitis was induced in male C57BL/6 mice using DSS5% for 5 days, control group received water. RNA was extracted from inflamed and non-inflamed colon. Using RT-qPCR, comparative analysis of 13 RGE was performed according to predefined criteria and relative colonic TNF-α and IL-1β gene expression was determined by calculating the difference in the threshold cycle.

Results: 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 variability within the inflamed and control groups. Conversely, TATA-box-binding protein (Tbp) and eukaryotic translation elongation factor 2 (Eef2) were not affected by inflammation and were the most stable genes. Normalization of colonic TNF-α and IL-1β mRNA levels was dependent on the reference gene used. Depending on the genes used to normalize the data, statistical significance varied from significant when TBP / Eef2 were used to non-significant when Gapdh, Actb or β2m were used.

Conclusions: This study highlights the appropriate choice of RGE to ensure adequate normalization of RT-qPCR data when using this model. Suboptimal RGE may explain controversial results from published studies. We recommend using Tbp and Eef2 instead of Gapdh, Actb or β2m as reference genes.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Confirmation of colitis induction by dextran sulphate sodium (DSS).
(A) Disease activity index, repeated measures ANOVA analysis followed by the multiple comparisons post hoc analysis; (B) macroscopic scores; (C) histological score. Control represents data obtained in non-colitic non-treated mice (n = 6/group). Student’s t-test analyses were used to compare DSS-treated group to the control group. Data are presented as the mean ± SD.
Fig 2
Fig 2. Systematic confirmation of colitis induction by dextran sulphate sodium (DSS).
(A) C-reactive protein serum level; (B) myeloperoxidase (MPO) activity in the colon; and colonic pro-inflammatory mediators: (C) TNF-α, (D) IL-6 and (E) IL-1β. Control represents data obtained in non-colitic non-treated mice (n = 6/group). Student’s t-test analyses were used to compare DSS-treated group to the control group. Data are presented as the mean ± SD.
Fig 3
Fig 3. Range of quantification/threshold cycle (Ct) values of the candidate reference genes.
A scatter dot plot displays the mean value and standard deviation in all colon samples (n = 12).
Fig 4
Fig 4. Effect of inflammation on candidate reference gene expression in colonic specimens.
DSS-induced colitic and non-inflamed control colon groups are defined in the Materials and Methods. Several genes were found to show altered expression between samples from control and DSS-induced colitis group (n = 6/group). Student’s t-test was used for comparison between the groups. Ct, threshold cycle.
Fig 5
Fig 5. The average expression stability values of the 13 reference genes analyzed by geNorm.
A lower M value refers to higher gene expression stability.
Fig 6
Fig 6. Reference gene stability performance of the 13 reference genes analyzed using BestKeeper.
Lower values refer to higher stability and higher values refer to a lower stability.
Fig 7
Fig 7. Comprehensive gene stability ranking for the 13 reference genes used in DSS-induced colitis and control groups.
RT-qPCR was performed for each of the 13 reference genes using the same RNA samples from control and DSS-induced colitis groups (inflamed and non-inflamed areas).
Fig 8
Fig 8
Effect of reference gene selection on the relative expression of colonic TNF-α (A) and IL-1β (B). Target gene expression was normalized against the 13 reference genes using comparative the ΔΔCt method. Significant differences between control and inflamed colon were seen only with the stable reference genes. Student’s t-test was used to compare the groups. Data is presented as the mean ± SD (n = 6/group).
Fig 9
Fig 9. Changes in the expression levels of 13 selected reference genes in colon of control and DSS treated groups.
The expression levels of these genes in colonic samples were normalized to the Ct values of ERCC-00113, an external control RNA. The graphs show relative expression values that calculated using the ΔΔCt method. Student’s t-test was used to compare the groups with significance level 0.05. Data are shown as mean ± S.D (n = 6 /group).
Fig 10
Fig 10. Illustration summary describes the effect of dextran sodium sulfate (DSS) on the reference genes expression (RGE) stability in the gastrointestinal mucosa and their impact when used to quantify mRNA level of target genes using RT-qPCR.
Moreover, the immune cellular composition can greatly vary and can contribute to the variation of expression of a given gene. The least stable genes are the suboptimal genes that showed high variability (Gapdh, Actb, β2m, Trfc), while the most stable genes are the optimal genes that exhibited high stability (Tbp, Eef2).
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Grants and funding

This study was supported by grants from Canadian Foundation for Innovation, Crohn’s and Colitis Canada, Research Manitoba, the Canadian Institutes of Health Research to JEG and the Children's Hospital Research Institute of Manitoba to NE and JEG.