Dextran sodium sulfate inhibition of real-time polymerase chain reaction amplification: a poly-A purification solution.

Abstract

BACKGROUND:

Dextran sulfate sodium (DSS) induces experimental colitis and promotes colitis-associated cancer in rodents. Here we document potent inhibition of real-time quantitative polymerase chain reaction (qPCR) using cDNA from DSS-exposed mouse tissues, which complicates gene expression analysis.

METHODS:

We characterize DSS inhibition of qPCR in-vitro and 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 real-time polymerase chain reaction amplification and gene expression analysis.

RESULTS:

DSS inhibits qPCR amplification of cDNA between 1 and 10 nM. Orally administered DSS interferes with qPCR amplification of cDNA derived from multiple tissues. Poly-A purification of DSS-exposed RNA allows reliable and cost-effective gene expression analysis in DSS-exposed tissue.

CONCLUSIONS:

DSS is a potent inhibitor of real-time qPCR amplification and interferes with tissue-specific gene expression analysis in DSS-exposed mice. Poly-A purification of tissue-derived RNA results in reliable and cost-effective gene expression analysis in DSS-exposed mice.

• Authors: T A Kerr, M A Ciorba, H Matsumoto, V R T Davis, J Luo, S Kennedy, Y Xie, A Shaker, B K Dieckgraefe, N O Davidson
• Journal: Inflammatory bowel diseases (Inflamm Bowel Dis) Vol. 18 Issue 2 Pg. 344-8 (Feb 2012) ISSN: 1536-4844 [Electronic] England
• PMID: 21618356 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2011 Crohn's & Colitis Foundation of America, Inc.

Chemical References

• DNA, Complementary
• RNA, Messenger
• Poly A
• RNA
• Dextran Sulfate

Topics

• Animals
• DNA, Complementary (antagonists & inhibitors, biosynthesis)
• Dextran Sulfate (administration & dosage, adverse effects)
• Gene Expression Profiling
• Mice
• Mice, Inbred C57BL
• Poly A (isolation & purification)
• RNA (isolation & purification)
• RNA, Messenger (isolation & purification)
• Real-Time Polymerase Chain Reaction (methods)