Dextran Sodium Sulfate Inhibition of Real-Time Polymerase Chain Reaction Amplification: A Poly-A Purification Solution

Abstract

Dextran sulfate sodium (DSS) induces experimental colitis and promotes colitis-associated cancer in rodents. In practice we observe potent inhibition of real-time quantitative PCR (qPCR) using cDNA from DSS-exposed mouse tissues, which complicates gene expression analysis. Here we characterize DSS inhibition of qPCR in a wide array of murine tissues following ingestion of DSS. We examine different approaches to RNA purification prior to cDNA synthesis in order to optimize cDNA amplification and gene expression analysis. We find that poly-A purification of DSS-exposed RNA allows cDNA synthesis and permits reliable and cost-effective mRNA quantification for gene expression analysis in DSS-exposed tissue.

Modeling Colitis-Associated Cancer with Azoxymethane (AOM) and Dextran Sulfate Sodium (DSS)

“…Because DSS itself can interfere with this process, we recommend poly-A purification prior to amplification. 22 Portions of colon can be flash frozen to be studied for protein expression by western blot. Investigators of colitis-associated cancer may be interested in visual markers of cell proliferation.…”

Lyn Deficiency Leads to Increased Microbiota-Dependent Intestinal Inflammation and Susceptibility to Enteric Pathogens

“…Tissue was homogenized in buffer RLT (Qiagen) using a gentleMACS tissue dissociator (Miltenyi Biotec). RNA was isolated using an RNeasy kit (Qiagen) according to the manufacturer's instructions, and genomic DNA was removed by treatment with DNAse I. Alternatively, RNA was isolated using TRIzol reagent (Sigma-Aldrich) followed by oligo(dT) purification of mRNA as previously described (25). Synthesis of cDNA was performed using an iScript cDNA synthesis kit.…”

Characterization of Colorectal Cancer Development in Apc min/+ Mice

“…In this chapter, we will describe techniques for studying colon cancer development in Apc min/+ mice on a C57BL/6J (B6) genetic background, focusing on Dextran Sulfate Sodium-induced (DSS) [14–17], high fat diet-induced [18–20], and bile acid supplementation [21, 22] as environmentally modifiable models of colorectal cancer. This chapter also includes protocols describing extraction and purification of DSS-contaminated RNA [23], as well as sampling, harvesting, and tissue processing. The common pathologic lesions encountered in these animals are described in detail.…”