U-STAR: Universal sequence-tag array technology for absolute quantification of per cell transcript profiles

基因表达分析的重要性和巨大的实用性现在已经得到了很好的确立，并且出现了两种主要技术：1)DNA微阵列提供了一种大规模平行和相对直接的方法，然而它们是基因组特异性的，可以提供相对的，但不是绝对的，每个细胞转录水平的定量；2)基因表达序列分析（SAGE）是一种基于测序的方法，可以提供绝对的转录物丰度数据，但由于高通量DNA测序中心的高成本和有限的可用性，以及昂贵、劳动密集型的样品制备，它处于严重劣势。该项目的目标是开发一种通用序列标签（微）阵列（U-STAR）平台，用于类似sage的分析，该平台结合了当前每种技术的优点并减轻了缺点。“通用”意味着单个阵列可以用于分析任何感兴趣的生物体或组织样本。该技术利用基于锁定核酸（LNA）的短探针微阵列，使我们能够设计每个探针-靶标双工的稳定性，从而平衡整个阵列的熔化温度。这反过来又允许杂交条件的识别，消除由于探针-目标不匹配的错误信号。一种新的检测系统也被提出，在检测极限和整体精度方面提供了比传统荧光标签潜在的优势。杂交U-STAR阵列的定量读数将通过开发一种完全集成的仪器来增强，该仪器采用纳米制造技术，在每个寄存器上提供高效、稳定和精确的测量。这项研究将产生新一代的DNA微阵列平台和相关仪器。区别于这项新技术的主要进步是：i)对任何感兴趣的生物体的普遍适用性；Ii)以每个细胞为基础提供真正定量分析的能力；Iii)集成仪器的速度、精度、便携性和节省成本的特点。因此，在研究和临床环境中，新技术将在众多生命科学和健康研究应用中具有价值。

The importance and tremendous utility of gene expression analysis is now well established and two principle technologies have emerged:  1) DNA microarrays offer a massively parallel and relatively straightforward approach, however they are genome specific and can provide relative, but not absolute, quantification of per cell transcript levels; 2) serial analysis of gene expression (SAGE) is a sequencing-based approach that can provide absolute transcript abundance data, however it is severely disadvantaged by high cost and limited availability of high-throughput DNA sequencing centres, and by costly, labor-intensive sample preparation.  The goal of this project is to develop a universal sequence-tag (micro)array (U-STAR) platform for SAGE-like analyses that combines the advantages, and mitigates the disadvantages, of each of the current technologies.  'Universal' means that a single array can be used to analyze any organism or tissue sample of interest.  The technology exploits short-probe microarrays based on locked nucleic acids (LNA) permitting us to engineer the stability of each probe-target duplex and thereby to equalize the melting temperature across the entire array.  This in turn allows the identification of hybridization conditions that eliminate false signals due to probe-target mismatches.  A new detection system is also proposed that offers potential advantages over conventional fluorescent tags in terms of detection limit and overall precision.  Quantitative reading of hybridized U-STAR arrays will be enhanced by developing a fully integrated instrument employing nanofabrication technologies to provide for highly efficient, stable and precise measurements at each register.  This research will yield a new generation of DNA microarray platform and related instrumentation.  The main advancements that distinguish this new technology are:  i) the universal applicability to any organism of interest; ii) the capability to provide truly quantitative analyses on a per cell basis; iii) the speed, precision, portability and cost saving features of the integrated instrumentation.  The new technology will thus be valuable in a multitude of life science and health research applications, both in research and clinical settings.