Abstract
BackgroundDextran sodium sulfate (DSS) is commonly used in mouse studies to induce a very reproducible colitis that effectively mimics the clinical and histological features of human inflammatory bowel disease (IBD) patients, especially ulcerative colitis. However, the mechanisms of action of DSS remain poorly understood, and observations by our laboratory and other groups indicate that DSS contamination of colonic tissues from DSS-treated mice potently inhibits the quantitative reverse-transcription polymerase chain reaction (qRT-PCR) amplification of mRNA.ResultsA prior study used poly-A-mediated mRNA purification to remove DSS from RNA extracts, but we herein report a second efficient and cost-effective approach to counteract this inhibition, using lithium chloride precipitation to entirely remove DSS from RNAs. We also explored how DSS interferes with qRT-PCR process, and we report for the first time that DSS can alter the binding of reverse transcriptase to previously primed RNA and specifically inhibits the enzymatic activities of reverse transcriptase and Taq polymerase in vitro. This likely explains why DSS-treated colonic RNA is not suitable to qRT-PCR amplification without a previous purification step.ConclusionIn summary, we provide a simple method to remove DSS from colonic RNAs, and we demonstrate for the first time that DSS can inhibit the activities of both polymerase and reverse transcriptase. In order to reliably analyze gene expression in the colonic mucosa of DSS-treated mice, the efficiency rate of qRT-PCR must be the same between all the different experimental groups, including the water-treated control group, suggesting that whatever the duration and the percentage of the DSS treatment, RNAs must be purified.
Highlights
Dextran sodium sulfate (DSS) is commonly used in mouse studies to induce a very reproducible colitis that effectively mimics the clinical and histological features of human inflammatory bowel disease (IBD) patients, especially ulcerative colitis
In vivo DSS exposure completely inhibits the qPCR process without altering RNA integrity To test whether in vivo DSS exposure can alter RNA integrity, WT mice were exposed to drinking water containing 3% DSS for 7 days
Agarose gel electrophoresis revealed that housekeeping gene 36B4 amplification products were obtained from control samples but not from DSS-treated samples (Figure 1B), demonstrating that the in vivo exposure of colonic tissue to DSS result in a complete inhibition of qPCR process
Summary
Dextran sodium sulfate (DSS) is commonly used in mouse studies to induce a very reproducible colitis that effectively mimics the clinical and histological features of human inflammatory bowel disease (IBD) patients, especially ulcerative colitis. Reported in 1985 by Ohkusa et al who used DSS to induce colitis in hamsters [3], the DSS model was thereafter extrapolated to mice [4]. This chemical compound is extensively used by investigators studying pathogenesis of colitis and factors affecting colitis. Colitis is induced by the addition of DSS to drinking water. The DSS model has been fully exploited for 20 years, the underlying mechanisms of DSS-induced colitis are not yet entirely understood
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