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

6 and NCIH716.7,8 Since GRP receptor antagonists inhibit the proliferation of these cell lines, the GRP receptor may represent a novel oncogene in adenocarcinomas of the colon. In order to determine whether GRP receptor expression is an early or late phenomenon in the development of colon cancer, we used RTISPCR to evaluate an adenomatous polyp removed from a human colon. Polyps are the earliest endoscopically visible lesions prior to the overt development of colon cancer and are therefore useful for evaluating the presence or absence of GRP receptor expression.

In situ PCR (ISPCR) allows the detection of DNA and RNA sequences in individual cells. Coupled with reverse transcription, RTISPCR permits the detection of rare mRNA species in their precise histological location and is superior to standard in situ hybridization.9,10 Furthermore, RTISPCR can be applied to archival tissues, specimens stored for years by pathology departments. Until now, such specimens have been an underutilized resource. Our technique is optimized for use on paraffin-embedded archival tissues and is modified from a number of different methodologies.11,12

RTISPCR Protocol

Pretreatment. Newly cut 5m slices are prepared from archival paraffin tissue blocks and mounted unstained on standard glass slides. Tissue sections are deparaffinized in three changes of xylene for 2 minutes each, followed by rehydration by exposing slides to successive 2-minute washes in graded ethanols (absolute, 95%, 70% and 50%). The slides are then treated with proteinase K (5 g/ml) in phosphate buffered saline (PBS) for 60 minutes at 37C in order to make the cells permeable. Following protease treatment, the slides are washed in PBS and then dehydrated in graded ethanols (50%, 70%, 95% and absolute) for 2 minutes each. These slices are treated with 100 l of 1 U/l DNase I, RNasefree, in a humidified chamber
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