DSS-mediated Inhibition of Quantitative Real-time Polymerase Chain Reaction

Abstract

The incidence of inflammatory bowel diseases (IBD), including ulcerative colitis (UC), is on the rise. UC is modeled in rodents by the consumption of dextran sodium sulfate (DSS). Quantitative real-time polymerase chain reaction (qPCR) is a sensitive technique used to quantify the abundance of “target gene sequences”, including those that may be related to the causation of UC. Using Lactobacillus-group-specific primers, qPCR was used to determine the abundance of Lactobacillus species found in the feces of both DSS-treated (n=4) and untreated control rats (n=6). DNA isolated from the feces of untreated control rats yielded strong and reproducible qPCR signals, whereas DNA isolated from the feces of DSS-treated rats often failed to produce detectable qPCR signals. We hypothesized that DSS co-purified with the extracted DNA and reduced PCR efficiency by either (i) competing with DNA for the Taq DNA polymerase active site or (ii) binding and modifying the polymerase. To test this hypothesis, we conducted DSS-spiking studies and employed a number of techniques to remove residual DSS. We confirm here that DSS is indeed responsible for the observed qPCR failure and that the Dneasy Blood and Tissue Kit (Qiagen) was most effective in removing residual DSS and restoring PCR efficiency.