Single-molecule FISH on a large scale requires advances on multiple fronts. First, chemical synthesis, labeling and purification of DNA oligos must be accomplished. Second, hybridization and imaging in a 96-well plate format must be developed. Finally, automated image analysis and classification must be implemented. We have begun collaborating with Biosearch to provide labeled DNA oligos to the laboratory. In the past, there was no commercial source for FISH probes, and all synthesis was done on a small scale in-house. Thus, having a partner with significant synthesis capabilities removes a major obstacle to high-throughput FISH analysis. Since February 2011, when The Systems Biology of Gene Expression Section in the National Cancer Institute was established, a custom fluorescence microscope was designed and built. This microscope will be optimized for single-molecule imaging and will also be capable of automated microscopy. In parallel, we have developed a new set of software tools for analyzing FISH data. These programs carry out spot-detection and segmentation along with cell segmentation. At present, we are testing the new algorithms on example data, but we anticipate this software will be made available to the scientific community. Single-cell approaches have indicated that gene expression varies considerably among cells in a population. Furthermore, there is a growing awareness that heterogeneity arises from the kinetics of transcription, which raises the question: how do cells maintain a coordinated expression program in response to stimuli? The estrogen response is rapid but it is impossible to ascertain from population measurements whether certain pathways show co-regulation in single cells. For diseases such as cancer, which arise from aberrations in single cells, these mechanisms of coordination may be critical to proper expression regulation. One possibility is that the nucleus is compartmentalized into regions where functionally-related genes are co-transcribed. We have measured nascent transcripts directly using single-molecule RNA-FISH to determine the extent of co-regulation of genes in response to estrogen. In addition, we have begun collaborative investigations on a variety of questions which are based on single-cell RNA analysis. A partial list of NIH collaborators using the FISH technique we have implemented includes: David Levens, Dinah Singer, Elissa Lei, and Bob Crouch.
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