Technical note: Reducing dextran sodium sulfate (DSS) interference with gene-expression quantification in a mouse model of colitis.

Abstract

Gene expression analysis via reverse transcription quantitative real-time polymerase chain reaction (qPCR) can be inhibited by various substances, including dextran sodium sulfate (DSS), a chemical commonly used to induce intestinal inflammation in animal models. Ensuring elimination and reduction of qPCR interference in tissues from laboratory animals following oral administration of DSS is critical and may improve the power of experimental tests. While methods for DSS removal have been reported, their effectiveness varies depending on the animal model, extraction techniques, DSS concentration, and treatment duration. We compared the effectiveness of the commonly used RNeasy Plus Universal Mini Kit with and without further lithium chloride (LiCl) precipitation at eliminating or reducing qPCR interference from RNA isolated from the colons of DSS-treated mice with histologically confirmed intestinal damage. The RNeasy Plus Universal Mini Kit alone was insufficient to eliminate interference in colonic tissue, as evidenced by increased quantification cycle (Cq) values and variation in the reference gene expression in DSS-treated distal colons (P adj. = 0.04). LiCl precipitation restored Cq values to control levels and reduced variation. DSS-treatment did not lead to interference in non-enteric tissues, including the spleen and cortex. LiCl precipitation not only restored reference gene expression but also improved the detection of changes in inflammatory markers, Il-6 and Tnf, in colonic tissue. In proximal colons, Il-6 was upregulated 2.97-fold in DSS-treated (non-LiCl precipitated, P adj. = 0.007), and 4.00-fold following LiCl precipitation (P adj. = 0.0004), compared to control mice. In distal colons, Il-6 was upregulated 3.95-fold in DSS-treated (non-LiCl precipitated, P adj. = 0.014) and 5.57-fold after LiCl precipitation (P adj. = 0.001) compared to control. Tnf was upregulated 2.05-fold in DSS-treated (non-LiCl precipitated, P adj. = 0.04), and 2.44 (P adj. = 0.009) after LiCl precipitation compared to controls in proximal colons. In distal colons, Tnf was 3.47-fold higher in DSS-treated (non-LiCl precipitated, P adj. = 0.009) and 4.41-fold higher after LiCl precipitation (P adj. = 0.002) compared to control. These findings demonstrate that the combined use of the RNeasy Plus Universal Mini Kit and LiCl precipitation enhances qPCR performance, ensuring reliable gene expression analysis in colonic tissue following acute DSS treatment in a murine model.