Platelet transcriptome analysis from small blood volumes

描述（由申请人提供）：人体血小板在正常的止血过程和病理状况（如血栓形成、血管重构、炎症和伤口修复）中发挥关键作用。血小板是由前体骨髓巨核细胞的细胞质芽产生的，虽然它们保留巨核细胞来源的mrna，但它们是无核的，缺乏核DNA。为了确定血小板转录组的分子解剖结构，我们采用了微阵列和基因表达序列分析（SAGE）的互补技术来对高度纯化的人类血小板（血液101:2285-2293）进行遗传分析，并证明了这种方法在罕见血小板疾病（原发性血小板减少症）分子分析中的潜在适用性。虽然这些观察结果建立了支持这一研究方向的初步原理证明，但目前的血小板分离程序需要血小板采集和相对繁琐的纯化方法来获得最佳测定，限制了更广泛的适用性。在此资助期间，我们建议开发一种小型化的高通量血小板转录组分析系统。在具体目标1中，我们将适应和发展机械剪切作为一种有效的方法，用于从全血（1ml）中分离超纯血小板，然后进行mRNA分离和具有代表性的转录物扩增，独特地适用于小RNA产量。在具体目标2中，我们将开发一种定制的血小板cDNA芯片，用于通过微阵列分析建立的扩增血小板mRNA保真度的验证性研究。如果成功，该项目将开发适当的基础设施和方法，以便对更大的数据集进行更全面的分析，这是该调查领域的一个主要长期目标。鉴于血小板在心血管疾病和中风中的重要性，这些研究将对新基因的发现和针对大量患者群体的分子诊断具有相当大的意义。

DESCRIPTION (provided by applicant): Human blood platelets play critical roles in normal hemostatic processes and pathologic conditions such as thrombosis, vascular remodeling, inflammation, and wound repair. Generated as cytoplasmic buds from precursor bone marrow megakaryocytes, platelets are enucleate and lack nuclear DNA, although they retain megakaryocyte-derived mRNAs. Towards the goal of defining the molecular anatomy of the platelet transcriptome, we have adapted complementary techniques of microarray and serial analysis of gene expression (SAGE) for genetic profiling of highly purified human blood platelets (Blood 101:2285-2293), and demonstrated the potential applicability of this approach for the molecular analysis of a rare platelet disorder (essential thrombocythemia). While these observations established the initial proof-of-principle supporting this research direction, current platelet isolation procedures require plateletpheresis and relatively cumbersome purification methods for optimal determinations, limiting wider applicability. During the tenure of this grant, we propose to develop a miniaturized system for high-throughput platelet transcriptome analysis. In specific aim 1, we will adapt and develop mechanical shear as an efficient methodology for separation of ultra-pure platelets from whole blood (1 mL), followed by mRNA isolation and representative transcript amplification uniquely adapted for small RNA yields. In specific aim 2, we will develop a customized platelet cDNA chip for confirmatory studies of amplified platelet mRNA fidelity as established by microarray analysis. If successful, this project will develop the appropriate infrastructure and methodologies for more comprehensive profiling of larger data sets, a major long-term goal of this area of investigation. Given the importance of platelets in cardiovascular disease and stroke, these studies will have considerable implications for novel gene discovery, and for molecular diagnostics targeted at large patient populations.