Parallel high sensitivity gene expression monitoring

描述（由申请人提供）：本提案的广泛，长期目标是开发一种新的高灵敏度微阵列技术，以便能够从非常有限的生物材料中准确检测弱表达基因。这将大大扩大DMA芯片技术的应用领域。具体目标是1)开发一个强大的协议，将微阵列的灵敏度提高7个数量级，与目前的技术相比，2)设计和验证一个用于生成扩增过程中使用的组合文库的标签序列文库，3)进行三个系列的实验来验证我们的方法并展示其广泛的应用范围。该项目与健康相关，为分子生物学家提供了一种独特的资源，可以从非常少量的起始材料中以无与伦比的灵敏度检测弱表达基因。这项新技术将极大地受益于许多关于关键基因表达水平低于当前检测极限的疾病发生和进展的研究。更好地了解这些疾病的分子机制无疑将加速新药的发现。我们的技术还将使开发新的分子诊断测试成为可能，这些测试与低侵入性活检技术提供的低材料兼容。研究计划包括三个阶段的放大。第一次扩增是在逆转录过程中，通过将基因特异性引物与报告序列结合，第二次扩增是通过对该报告序列进行RNA扩增，最后一次扩增是通过基于金属纳米粒子共振光散射特性的高灵敏度信号检测。实验方法将包括开发用特定标签标记mRNA的技术，并用T7 RNA聚合酶扩增这些标签。随后将用金和银颗粒间接标记，这比荧光染料产生更高的信号。T7扩增后的标记序列集合将通过杂交进行验证。使用更少的材料重复已发表的实验将验证我们的技术。进一步的验证将通过进行新的实验来检测参与脂肪形成的弱表达基因，并监测受感染宿主细胞中的细菌基因表达。

DESCRIPTION (provided by applicant): The broad, long-term objectives of this proposal are to develop a new highly sensitive micro array technology to enable the accurate detection of weakly expressed genes from very limited amounts of biological material. This will enlarge the field of application of the DMA chip technology significantly. The specific aims are 1) to develop a robust protocol to increase micro array sensitivity by seven orders of magnitude compared to current technology, 2) to design and validate a library of tag sequences used to generate a combinatorial library to be used in the amplification process and 3) to conduct three series of experiments to validate our approach and demonstrate its broad range of application. The health relatedness of this project is to provide molecular biologists with a unique resource to detect weakly expressed genes with an unmatched sensitivity from a very low amount of starting material. Numerous studies on the initiation and progression of diseases where key genes are expressed at levels below current detection limit will greatly benefit from this new technology. A better understanding of molecular mechanisms involved in these diseases will undoubtedly speed up the discovery of new drugs. Our technology will also make possible the development of new molecular diagnosis tests compatible with the low amount of material available from the lesser invasive biopsy techniques. The research plan consists of three stages of amplification. The first amplification is during the reverse transcription by using gene specific primers combined to a reporter sequence, the second amplification is by doing an RNA amplification of this reporter and the final amplification is by using a high-sensitivity signal detection based on the resonance light scattering properties of metal nanoparticles. The experimental approach will involve the development of techniques to label mRNA with specific tags and amplify these tags with the T7 RNA polymerase. This will be followed by an indirect labeling with gold and silver particles, which generate a higher signal than fluorescent dyes. A collection of tag sequences will be validated by hybridization after T7 amplification. Repeating a published experiment using a much lower amount of material will validate our technology. Further validation will be by conducting new experiments to detect weakly expressed genes involved in adipogenesis, and to monitor bacterial gene expression in infected host cells.