Multiplex arrays using Confocal Raman for BRCA1 alternative splice profiling

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DESCRIPTION (provided by applicant): We will elucidate alternative splice (AS) profiles in nontumorigenic Human breast epithelial cell (HBEC) lines and compare these profiles to those obtained from tumorigenic cell lines to evaluate whether BRCA1 AS variants are specific to pathologic conditions (e.g., a malignant phenotype). The key requirement of AS profiling is to detect large number of AS variants in the mRNA pool of the target gene. Because of the overlap of the broad (300-400 nm wide) fluorescent bands only 3-4 tags could be simultaneously handled by the conventional microarray technology, limiting the AS variants that can be investigated to 4 or less. Consequently only the four predominant AS variants of BRCA1 have been extensively studied so far. In the proposed Confocal Raman spectroscopy/imaging approach, several orders of magnification are possible due to the resonance and enhancement effect and distinct vibrational mode peaks at 1 nm resolution is possible. Considering the choice or tags available (over 1000), practically an unlimitted number of tagged AS variants could be used to explore the AS variants, thus providing limitless possibility. Long-term goal is to develop a confocal raman-based work station for ~$100k complete with data analysis and image processing capability to monitor up to 10 different interactions in a 1 micron diameter spot, by careful choice of labels and nanoparticles for enhancement along with an appropriate array fabrication protocol. Specific aims of this research are to (1) synthesize Raman-labeled oligonucleotide probes to capitalize on the magnification due to the resonance effect and surface enhancement due to gold particles for the five selected raman tags (fluorescent and nonfluorescent tags), (2) fabricate - 100, 50, and 1 urn diameter spot size arrays and develop a Confocal raman imaging method using appropriate lasers excitations corresponding to the five selected tags to monitor up to five different interactions in a single spot, and finally (3) test the multiplex concept to effectively elucidate AS patterns of the BRCA 1 gene from the two chosen cell types together with relevant data interpretation methods. Deliverables include, appropriate choice of raman tags for desired amplification with an optimum combination of gold particle, array fabrication protocol, tests on assay sensitivity and specificity, detection of 5 different interactions on a single spot and examination of the AS variants of the chosen cells.