Open AccessDextran sodium sulfate inhibition of real‐time polymerase chain reaction amplification: A poly‐A purification solution
Author(s) -
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.
Publication year - 2012
Publication title -
inflammatory bowel diseases
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.932
H-Index - 146
eISSN - 1536-4844
pISSN - 1078-0998
DOI - 10.1002/ibd.21763
Subject(s) - microbiology and biotechnology , real time polymerase chain reaction , gene expression , complementary dna , polymerase chain reaction , chemistry , rna extraction , rna , gene , biology , biochemistry
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;)