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 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
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 overnight at 37C. (We place the slides and a moist paper towel in a sealed plastic container in an incubator.) The next day, slides are washed by immersion in PBS for 5 minutes and dehydrated in graded ethanols as described.
Reverse transcription. RNA is reverse transcribed to cDNA in a 40l mixture containing 20 mM Tris-HCL (ph 8.4), 50 mM KCl, 2.5 mM MgCl2, 0.2 mM of each deoxynucleotide triphosphate (dNTP), 100 ng of selected reverse primer (we use 20 mers) and 2 U of reverse transcriptase at 42C for 45 minutes in a humidified chamber. Following reverse transcription, the slides are washed by immersion in PBS and dehydrated in graded ethanols as described.
PCR amplification. PCR amplification is performed in a 100l mixture containing 20 mM Trishcl (ph 8.3), 50 mM kcl, 1.5 mM MgCl2, 0.001% gelatin, 0.2 mM dntps, 15% glycerol and 100 ng of each primer. Primers should be designed to yield a product less than 300 bp in size, and we use the same reverse primer as used for reverse transcription. This reaction mixture is applied to each tissue slice and covered using the resealable GeneFrame product. Amplification is then performed using the in situ adapter for the RoboCycler 40 Temperature Cycler. The following conditions are used: 96C for 2 minutes, 40 cycles at 96C for 30 seconds, 52C for 30 seconds, 74C for 60 seconds and one cycle at 74C for 5 minutes. After cycling, the slices are washed by immersion for 5 minutes in PBS and then dehydrated in graded ethanols as described.
Hybridization. The probe is readily generated by PCR using digoxigeninlabeled primers. In situ PCR amplification products are then detected by hybridization to digoxigeninlabeled DNA probes9 in hybridization buffer (2x SSC, 50% formamide, 7.5% dextran sulfate and 100 to 200 ng of probe). The hybridization solution is added to the slices moun ted on the slides, and the slides are sealed and heated to 95C for 15 minutes and maintained at 37C overnight for hybridization. The next day, the hybridized slides are washed in 2.5% BSA in 0.2x SSC for 15 minutes at 42C, followed by PBS for 5 minutes. After washing, 100 l of a 1:200 dilution of alkaline phosphataseconjugated, antidigoxigenin antibody (BoehringerMannheim, Indianapolis, IN) are added to the slides, and the slides are incubated for 1 hour in a humidified chamber. The slides are then washed in PBS for 5 minutes and developed with NBT/BCIP solution. The reaction is stopped by washing in PBS, followed by dehydration of the tissue in graded ethanols (figure 2). The specimens are covered using Situ/Mount product. We avoid standard mounts because they can solubilize the stain, causing it to disappear over time.
Controls. All specimens should be processed in triplicate, allowing for both positive and negative controls. The negative control is processed as described except that reverse transcriptase is excluded from the reaction mixture; the positive control slide is not treated with DNase I. The RoboCycler 40 Temperature Cycler in situ adapter system conveniently holds three standard microscope slides so that the experimental tissue can be processed simultaneously with positive and negative controls (data not shown).
Caveats. Subjective interpretation of immunohistochemistry to determine the number of positive cells should be avoided. Rather, an objective approach, such as quantitative PCR,13 should be used. Quantitative PCR can be performed on RNA extracted from a 20-M sample of freshly sectioned archival tissue.14 Finally, if counter staining of the tissue is necessary, it is important to use a stain that will not be solubilized by the Situ/Mount product, such as BC50 Red stain.
RTISPCR is a recently developed approach for detecting rare s that often cannot be detected by traditional approaches, including in situ hybridization. RTISPCR can be used on archival tissues of almost any age, thereby giving new life to the samples maintained by pathology departments. In this study, we used RTISPCR to identify the expression of GRP receptors by mucosal epithelial cells in a human colonic polyp (figure 2). Our results suggest that expression of the GRP receptor may occur early in the development of colon cancer.
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