Platelet transcriptome analysis from small blood volumes

描述（由申请人提供）：人血小板在正常止血过程和病理状态（如血栓形成、血管重塑、炎症和伤口修复）中起关键作用。血小板是由骨髓巨核细胞的前体细胞质芽产生的，虽然它们保留了巨核细胞衍生的mRNA，但它们是去核的，缺乏核DNA。为了定义血小板转录组的分子解剖学的目标，我们已经采用了基因表达的微阵列和系列分析（SAGE）的互补技术用于高度纯化的人血小板的遗传分析（Blood 101：2285-2293），并证明了这种方法用于罕见血小板病症（原发性血小板增多症）的分子分析的潜在适用性。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去除术和相对繁琐的纯化方法才能进行最佳测定，从而限制了更广泛的适用性。在此资助的任期内，我们建议开发一个小型化的高通量血小板转录组分析系统。在具体目标1中，我们将调整和开发机械剪切作为从全血（1 mL）中分离超纯血小板的有效方法，然后进行mRNA分离和代表性转录本扩增，这是唯一适合小RNA产量的方法。在具体目标2中，我们将开发定制的血小板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。考虑到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产生重大影响。

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.