Analysis of gene expression at the RNA level often constitutes a first important step in the characterization of a novel gene. In addition, when using marker genes in this type of analysis molecular and cellular changes can be discovered that may occur in mutant and treated tissues. In contrast to immunohistochemical analyses that require a suitable antibody, RNA In situ hybridization can be readily carried out for neariy any gene of interest. Although ISH is a widely used method, the effort to introduce this technique into each of the BCM-IDDRC laboratories would be considerable and very costly. Thus having a core unit that uses existing and highly sophisticated equipment provides not only cost saving but also ensures efficiency and high quality of data. In situ hybridization has been a method used extensively in various fields of developmental biology. The conventional, manual method allows relatively few slides (usually 10 -20) to be processed at once, and the whole experiment can take at least 3 days plus the exposure time (up to several weeks) when radioactively labeled riboprobes are used. The novel aspect of our in situ core is that we use standardized methods for tissue preparation, tissue sectioning, probe preparation, in situ hybridization and data collection. Our robotic ISH is performed in special flow through hybridization chambers that are fitted into temperature controlled chamber racks on the platform of a Tecan pipetting robot. All solutions (including riboprobes) are delivered by the pipetting robot to the hybridization chambers. This allows us to do many more wash steps compared to using a manual set up. We find that a relatively short (6 hrs) hybridization time gives better results than the overnight incubations used with manual ISH. With short hybridizations, we have the capacity to process up to 192 slides in 20 hours. This means during a normal work-week we can process more than 750 slides. We also have a automated microscope that converts the expression patterns on the slides into electronic files that can be send over the internet and also posted on GenePaint.org, the web site shared with our collaborators at the Max Planck Institute in Germany. We have also developed software (GeneDetect) that allows us to quantify in situ results to help with comparison studies for mutant and wild type tissue or treated and untreated tissue. This standardization of methods together with our vast expertise in handling very different types of tissue enable us to perform these ISH experiment in the most efficient way possible and to obtain excellent results for all the different projects. As noted above, we can process 192 slides in one single ISH run which allows the ability to compare gene expression in sections from animals with different genotypes and/or different treatments hybridized under the same conditions, increasing uniformity of results. Clearly, it would be very difficult and time consuming for individual labs to set up ISH as a standard procedure.
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