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

基本信息

批准号：
7198004
负责人：
Joseph MK Irudayaraj
金额：
$ 7.4万
依托单位：
PURDUE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-04-01 至 2009-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7198004
关键词：
Alternative Splicing BRCA1 gene Biological Assay Breast Caliber Cell Line Cells Complex Condition Data Data Analyses Detection Electromagnetics Electronics Epithelial Cells Gene Targeting Genes Goals Gold Human Image Label Lasers Ligation Mammary Neoplasms Mediating Messenger RNA Methods Microarray Analysis Mission Molecular Molecular Profiling Monitor Nature Numbers Oligonucleotide Probes Oligonucleotides Pathologic Pattern Protein Isoforms Protocols documentation RNA Splicing Raman Spectrum Analysis Reporting Research Resolution Sampling Sensitivity and Specificity Spottings Structure Surface System Techniques Technology Testing Transcript Variant Work base cell type concept design desire fluorophore image processing innovation malignant phenotype nanoparticle neoplastic cell new technology particle single molecule size tumorigenic

项目摘要

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）：我们将阐明非致瘤性人乳腺上皮细胞 (HBEC) 系中的选择性剪接 (AS) 谱，并将这些谱与从致瘤性细胞系中获得的谱进行比较，以评估 BRCA1 AS 变体是否对病理状况具有特异性（例如，乳腺癌）。，恶性表型）。 AS 分析的关键要求是检测目标基因 mRNA 库中的大量 AS 变异。由于宽（300-400 nm 宽）荧光带的重叠，传统微阵列技术只能同时处理 3-4 个标签，从而将可研究的 AS 变体限制为 4 个或更少。因此，迄今为止仅对 BRCA1 的四种主要 AS 变体进行了广泛研究。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，几个数量级的放大倍数是可能的，并且在 1 nm 分辨率下可能出现不同的振动模式峰值。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基于共焦拉曼的工作站，价格约为 10 万美元，具有数据分析和图像处理功能，可通过仔细选择标签和纳米粒子来增强，以监测 1 微米直径点中多达 10 种不同的相互作用。使用适当的阵列制造协议。这项研究的具体目标是（1）合成拉曼标记的寡核苷酸探针，以利用共振效应和金颗粒表面增强的放大作用，用于五个选定的拉曼标签（荧光和非荧光标签），（2）制造- 100、50 和 1 瓮直径的光斑尺寸阵列，并使用与五个选定标签相对应的适当激光激发来开发共焦拉曼成像方法，以监测多达五种不同的相互作用最后 (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 AS 模式以及相关的数据解释方法。可交付成果包括：适当选择用于所需扩增的拉曼标签以及金颗粒的最佳组合、阵列制造方案、测定灵敏度和特异性测试、单个点上 5 种不同相互作用的检测以及所选细胞的 AS 变体的检查。