Multiplex arrays using Confocal Raman for BRCA1 alternative splice profiling

描述（由申请人提供）：我们将阐明非致瘤性人乳腺上皮细胞（HBEC）系中的可变剪接（AS）谱，并将这些谱与从致瘤性细胞系获得的谱进行比较，以评估BRCA 1 AS变体是否对病理条件（例如，恶性表型）。AS谱分析的关键要求是检测靶基因mRNA库中的大量AS变体。由于BRCA 1的宽荧光带重叠（300-400 nm宽），传统的微阵列技术只能同时处理3-4个标记，限制了可研究的AS变体为4个或更少，因此迄今为止，只有4种主要的BRCA 1 AS变体得到了广泛的研究。在所提出的共焦拉曼光谱/成像方法中，由于共振和增强效应，几个放大倍数是可能的，并且在1 nm分辨率下的不同振动模式峰是可能的。考虑到可用的选择或标签（超过1000个），实际上可以使用无限数量的标记的AS变体来探索AS变体，从而提供无限的可能性。长期目标是开发一个共焦拉曼工作站，约10万美元，完成数据分析和图像处理能力，以监测多达10个不同的相互作用，在1微米直径的斑点，通过仔细选择标签和纳米粒子的增强沿着与适当的阵列制造协议。本研究的具体目的是（1）合成拉曼标记的寡核苷酸探针，以利用由于共振效应而产生的放大和由于金颗粒而产生的表面增强，用于所选的五种拉曼标记物（荧光和非荧光标记），（2）制造-100，50，和1 μ m直径的光斑尺寸阵列，并开发共焦拉曼成像方法，其使用对应于五个所选标签的适当激光激发来监测多达五个最后（3）测试多重概念，以有效地阐明来自两种所选细胞类型的BRCA 1基因的AS模式以及相关数据解释方法。可验证的包括，适当选择用于所需扩增的拉曼标记与金颗粒的最佳组合、阵列制造方案、测定灵敏度和特异性的测试、在单个点上检测5种不同的相互作用以及检查所选细胞的AS变体。

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.