Multiplex arrays using Confocal Raman for BRCA1 alternative splice profiling

描述(申请人提供)：我们将阐明非致癌人类乳腺上皮细胞(HBEC)系中的选择性剪接(AS)谱，并将这些谱与从致瘤细胞系中获得的谱进行比较，以评估BRCA1作为变异体是否对病理条件(例如，恶性表型)具有特异性。AS图谱的关键要求是在靶基因的mRNA库中检测到大量的AS变异体。由于宽(300-400 nm宽)荧光带的重叠，传统的微阵列技术只能同时处理3-4个标签，从而将可以研究的AS变体限制在4个或更少。因此，到目前为止，只对BRCA1的四个主要变体进行了广泛的研究。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，可能有几个数量级的放大，并且可能在1 nm分辨率下有明显的振动模峰。考虑到可供选择的标签(超过1000个)，实际上可以使用无限数量的标记为变体来探索AS变体，从而提供了无限的可能性。长期目标是开发一种基于共聚焦拉曼的工作站，耗资约10万美元，具备数据分析和图像处理能力，通过仔细选择用于增强的标签和纳米颗粒以及适当的阵列制造协议，在1微米直径的光斑中监控多达10种不同的相互作用。本研究的具体目标是：(1)合成拉曼标记的寡核苷酸探针，以利用所选的五种拉曼标签(荧光和非荧光标签)由于金粒子的共振效应和表面增强而产生的放大效应；(2)制作-100、50和1 nm直径的斑点阵列，并利用与所选的五种标签相对应的适当激光激发来发展一种共聚焦拉曼成像方法，以在一个点上监测多达五种不同的相互作用；以及(3)测试多重概念，以有效地阐明两种所选细胞类型的BRCA 1基因的模式以及相关的数据解释方法。交付内容包括：为所需的扩增选择适当的拉曼标签和最佳的金粒子组合、阵列制造方案、检测灵敏度和特异性测试、在单个点检测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.