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很好的确立，并且已经出现了两种主要技术：1）DNA微阵列提供了大规模平行和相对简单的方法，然而它们是基因组特异性的，并且可以提供相对的，但不是绝对的，定量每个细胞转录物水平; 2）基因表达系列分析（SAGE）是一种基于测序的方法，可以提供绝对的转录本丰度数据，但由于高成本和高通量DNA测序中心的有限可用性，以及昂贵的劳动密集型样品制备。 该项目的目标是开发一个通用的序列标签（微）阵列（U-STAR）平台，用于SAGE样分析，结合了当前每种技术的优点，并减轻了缺点。 “通用”意味着单个阵列可用于分析任何感兴趣的生物体或组织样品。 该技术利用基于锁核酸（LNA）的短探针微阵列，使我们能够设计每个探针-靶双链体的稳定性，从而使整个阵列的解链温度相等。 这又允许鉴定杂交条件，其消除由于探针-靶错配引起的假信号。 还提出了一种新的检测系统，该系统在检测限和整体精度方面比传统荧光标记具有潜在的优势。 将通过开发采用纳米纤维技术的完全集成的仪器来增强杂交U-STAR阵列的定量阅读，以在每个寄存器处提供高效、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。 区分这种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）在每个细胞的基础上提供真正定量分析的能力; iii）集成仪器的速度，精度，便携性和成本节约功能。 因此，这项新技术将在研究和临床环境中的众多生命科学和健康研究应用中发挥重要作用。

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.