Platelet transcriptome analysis from small blood volumes

描述(申请人提供)：人的血小板在正常的止血过程和病理状态中起着关键作用，如血栓形成、血管重塑、炎症和伤口修复。血小板是由前体骨髓巨核细胞产生的细胞质芽，是去核的，没有核DNA，尽管它们保留了来自巨核细胞的mRNAs。为了确定血小板转录组的分子解剖学的目标，我们采用了互补的微阵列和基因表达序列分析(SAGE)技术来对高纯度的人血小板(血液101：2285-2293)进行遗传图谱分析，并证明了这种方法在一种罕见的血小板疾病(特发性血小板增多症)的分子分析中的潜在适用性。虽然这些观察确立了支持这一研究方向的初步原则证明，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才能获得最佳检测结果，从而限制了更广泛的适用性。在这笔赠款的任期内，我们建议开发一种用于高通量血小板转录组分析的微型系统。在具体目标1中，我们将采用和开发机械剪切法作为从全血(1毫升)中分离超纯血小板的有效方法，然后进行mRNA分离和代表性转录扩增，以获得独特的小RNA产量。在特定的目标2中，我们将开发一种定制的血小板基因芯片，用于通过微阵列分析建立的扩增的血小板mRNA保真度的确证研究。如果成功，该项目将开发适当的基础设施和方法，以便更全面地分析更大的数据集，这是这一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，这些研究将对新的基因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.