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 the initial parts were purchased. 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. During the summer of 2011, Celine Hong was a summer intern in the section. During her time in the lab, she carried out a meta-analysis on two recently published data sets on breast cancer gene regulation. This work leads to direct predictions about the functional significance of co-regulation of genes during estrogen signaling. The testable hypotheses generated from this bioinformatic analysis will form the basis of several experimental efforts in the section in the next fiscal year. For example, we are designing a library of FISH probes to look at genes whose expression is linked to breast cancer. Some of the questions we will be addressing include: how frequently are these genes expressed in the population? Are genes involved in particular pathways co-expressed at the level of single cells? How much noise exists in the expression of these genes, and does this noise effect the process of tumorigenesis? When genes are up-regulated during breast cancer, are they uniformly up-regulated, or do certain cells exhibit strong responses while other cells exhibit weak responses?
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