Reducing dextran sodium sulfate interference with gene expression quantification in a mouse model of colitis

Area of Science:

• Molecular Biology
• Genomics
• Immunology

Background:

• Dextran sodium sulfate (DSS) is widely used to induce experimental intestinal inflammation in animal models.
• DSS can interfere with gene expression analysis, particularly quantitative real-time polymerase chain reaction (qPCR), affecting experimental outcomes.
• Accurate RNA isolation and elimination of inhibitors are crucial for reliable gene expression studies in DSS-treated tissues.

Purpose of the Study:

• To evaluate the effectiveness of lithium chloride (LiCl) precipitation in removing DSS-induced qPCR interference from mouse colonic RNA.
• To compare the performance of a standard RNA extraction kit with and without LiCl precipitation.
• To assess the impact of LiCl precipitation on the accurate measurement of inflammatory gene expression.

Main Methods:

• RNA was isolated from colonic tissues of DSS-treated and control mice using the RNeasy Plus Universal Mini Kit.
• A subset of samples underwent additional lithium chloride (LiCl) precipitation.
• Quantitative real-time polymerase chain reaction (qPCR) was performed to analyze reference gene expression and inflammatory markers (Il-6, Tnf).

Main Results:

• The standard RNeasy Plus Universal Mini Kit alone was insufficient to eliminate DSS interference, showing increased quantification cycle (Cq) values and reference gene variation.
• LiCl precipitation restored Cq values to control levels and reduced variation in colonic RNA.
• LiCl precipitation improved the detection of DSS-induced upregulation of inflammatory markers Il-6 and Tnf in colonic tissues.

Conclusions:

• Combining the RNeasy Plus Universal Mini Kit with LiCl precipitation is essential for reliable gene expression analysis in DSS-treated mouse colonic tissue.
• LiCl precipitation effectively eliminates DSS interference, enhancing qPCR accuracy and the detection of inflammatory responses.
• This optimized method improves the power of experimental tests involving DSS-induced intestinal inflammation.