Multiplex arrays using Confocal Raman for BRCA1 alternative splice profiling

使用共焦拉曼进行 BRCA1 替代剪接分析的多重阵列

• 批准号: 7100387
• 负责人: Joseph MK Irudayaraj
• 金额: $ 7.62万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7100387
• 关键词: cell line; cell type; choice; conditioning; gene targeting; gold; human; mammary gland; microarray technology; oligonucleotides; particle; phenotype

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.

描述（由申请人提供）：我们将阐明非瘤性人乳腺上皮细胞（HBEC）系中的替代剪接（AS）曲线，并将这些剖面与从肿瘤细胞系获得的曲线进行比较，以评估BRCA1作为变体是否特定于病理条件（例如，恶性表型）。 AS分析的关键要求是检测靶基因的mRNA库中的大量AS变体。由于传统的微阵列技术可以同时处理宽（300-400 nm宽）荧光带的重叠（300-400 nm宽），只能同时处理3-4个标签，从而限制了可以研究4或更少的变体。因此，到目前为止，仅对BRCA1的变体进行了广泛研究。在提出的共聚焦拉曼光谱/成像方法中，由于共振和增强效果以及在1 nm分辨率下的不同振动模式峰，因此可以进行几种放大率。考虑到可用的选择或标签（超过1000个），实际上可以使用无限制的标记为变体，可用于探索AS变体，从而提供无限的可能性。长期目标是开发一个基于共焦拉曼的工作站，以约100k美元的价格提供数据分析和图像处理能力，以通过仔细选择标签和纳米粒子以及适当的阵列制造协议来监视1微米直径斑点中多达10种不同的相互作用。 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对于五个选定的标签，可以在单个位置监视多达五个不同的相互作用，最后（3）测试多重概念，从两个选择的细胞类型以及相关的数据解释方法有效地阐明为BRCA 1基因的模式。可交付成果包括，适当的选择拉曼标签，以进行所需的扩增，最佳组合金粒子，阵列制造协议，测定敏感性和特异性的测试，在单个点上检测5种不同的相互作用并检查所选单元的变体。