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

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...

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Bibliographic Details
Published in:Inflammatory bowel diseases 2012-02, Vol.18 (2), p.344-348
Main Authors: Kerr, T.A., Ciorba, M.A., Matsumoto, H., Davis, V.R.T., Luo, J., Kennedy, S., Xie, Y., Shaker, A., Dieckgraefe, B.K., Davidson, N.O.
Format: Article
Language:English
Subjects:
Animals
colitis
colitis‐associated cancer
dextran sodium sulfate
Dextran Sulfate - administration & dosage
Dextran Sulfate - adverse effects
DNA, Complementary - antagonists & inhibitors
DNA, Complementary - biosynthesis
Gene Expression Profiling
Mice
Mice, Inbred C57BL
Poly A - isolation & purification
qPCR
Real-Time Polymerase Chain Reaction - methods
RNA - isolation & purification
RNA, Messenger - isolation & purification
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Summary: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. (Inflamm Bowel Dis 2011;)
ISSN:1078-0998
1536-4844
DOI:10.1002/ibd.21763