Increasingly, there has been a realization of the importance of accurate assessment of chromatin modifications for both clinical care (for example for cancer detection, prognosis, determining treatment, designing new therapies and predicting response) as well as for understanding carcinogenesis. Currently available approach for identification of specific genome-associated proteins or DNA modifications, the chromatin Immunoprecipitation (ChIP) assay, is of limited clinical utility given that it requires time consuming/labor intensive pre-analytical sample preparation, provides low throughput, and is associated with epitope loss (raising concerns about data accuracy/reproducibility). To address some of these issues, we developed the Matrix ChIP, a microplate-based method which accelerates the analytical process, and increases assay's throughput, sensitivity and reproducibility. We now (Aims#1&2) propose to develop next generation microplate based platform, PIXUL-ChIP, which will integrate cancer sample preparation/ chromatin fragmentation with immunoprecipitation. PIXUL-ChIP will provide high throughput, simultaneous, and automated analysis of a wide range of chromatin marks which will allow us to dramatically expand our understanding of the role of chromatin alterations in health and disease. Current approaches to collect and prepare clinical specimens are not optimized for chromatin analysis. To assure the success of the PIXUL-ChIP, in Aim&3 we will develop and validate methods for collection, fixation, storage and ultrasound treatment of human normal and cancer (tissues/fluid) specimens. Specifically we propose:
Aim#1. To build a pixelated ultrasound (PIXUL) processor for fast and high-throughput chromatin fragmentation. We will utilize our recently pioneered ultrasound technology to build a PIXUL microplate processor for cell culture and tissue chromatin fragmentation in 96 well plates.
Aim#2. To combine sample preparation/chromatin with immunoprecipitation into an integrated microplate platform for high throughput epigenetic analysis, PIXUL-ChIP. The ability to shear chromatin in a microplate will streamline sample preparation, immunoprecipitation and ChIP DNA analysis.
Aim#3. Using PIXUL technology we will develop and validate practical cost-effective approaches for sample collection from human cancer tissues and their preparation for analysis of epigenetic alterations. Sample preparation and storage conditions dramatically affect the epitope loss, thus determining how to best collect/process/store biospecimens is of great importance. To this end we will develop protocols for sample collection/ preparation/storage and analysis for snap-frozen fresh and for formalin fixed paraffin embedded (FFPE) tissues. The user-friendly, high sensitivity/throughput PIXUL-ChIP technology will greatly increase the utility of new and archived cancer biospecimens and will facilitate basic and translational research to exploit epigenetic information from the vastly unexplored human cancer biospecimes.
Increasingly, there has been a realization of the importance of accurate assessment of chromatin modifications for both clinical care (for example, for cancer detection, prognosis, determining treatment, designing new therapies, and predicting response) as well as for understanding carcinogenesis. A strategy is proposed to develop tissue sample harvesting, storage and processing methods to maximize cancer biospecimens' utility for high throughput epigenetic analysis using a novel PIXUL-ChIP platform. These pre-analytical and analytical tools will advance discovery, validation of cancer biomarkers improving clinical outcomes in patients with malignancies.
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