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Detecting Gastrin-Releasing Peptide Receptor by in situ PCR on Archived,,,Tissue

In situ Adapter for the RoboCycler 40 Temperature Cycler allows rapid amplification of rare mRNA

Donald D. Lorimer * Robert E. Carroll * Richard V. Benya
Department of Medicine, University of Illinois at Chicago

In situ adapter system for the RoboCycler 40 temperature cycler is designed for holding standard microscope slides.

Reverse transcriptase in situ PCR (RTISPCR) is a recently described method for detecting mRNAs in archival paraffin tissue blocks. This method is readily performed using the RoboCycler 40 Temperature Cycler# and its microscope slide attachment, the in situ Adapter for the RoboCycler 40 Temperature Cycler. In this article, we describe our RTISPCR method for detecting the rare message for the gastrin-releasing peptide (GRP) receptor expressed by a previously resected gastrointestinal tract tumor. By detecting mRNAs in tissues that have been maintained in paraffin blocks and archived for over 50 years, this technology promises to dramatically expand the use of PCR amplification.

The GRP receptor is a member of the 7-transmembranespanning, G protein-coupled receptor superfamily. Within the gastrointestinal (GI) tract, GRP receptors are normally expressed by smooth muscle cells and by nerve endings, acting to regulate intestinal motility and to induce the secretion of other enteric peptide hormones.1-3 However, GRP receptors are not expressed by mucosal epithelial cells lining the normal GI tract in the few mammals studied, including dogs4 and humans.5 In contrast, recent studies have shown that GRP receptors are aberrantly expressed by a number of human colon cancers6 and colon cancer cell lines including HT29, MC2
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