Platelet transcriptome analysis from small blood volumes

小血容量的血小板转录组分析

• 批准号: 6763729
• 负责人: Dmitri V GNATENKO
• 金额: $ 20.69万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6763729
• 关键词: biotechnology; blood volume; clinical research; fluorimetry; gene expression; high throughput technology; human subject; messenger RNA; method development; microarray technology; nucleic acid amplification techniques; nucleic acid hybridization; nucleic acid purification; nucleic acid quantitation /detection; platelets; polymerase chain reaction; serial analysis of gene expression; biotechnology; blood volume; clinical research; fluorimetry; gene expression; high throughput technology; human subject; messenger RNA; method development; microarray technology; nucleic acid amplification techniques; nucleic acid hybridization; nucleic acid purification; nucleic acid quantitation /detection; platelets; polymerase chain reaction; serial analysis of gene expression

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.

描述（由申请人提供）：人血小板在正常的止血过程和病理状况（例如血栓形成，血管重塑，炎症和伤口修复）中起关键作用。血小板是由前体骨髓巨核细胞的细胞质芽产生的，是粉状的，缺乏核DNA，尽管它们保留了巨核细胞衍生的mRNA。为了定义血小板转录组的分子解剖结构，我们已经适应了微阵列的互补技术和基因表达的序列分析（SAGE），用于高度纯净的人类血小板（血液101：2285-2293）的基因分析（SAGE），并证明了这种方法对稀有小音调的潜在适用于这种方法。尽管这些观察结果确定了支持该研究方向的初始原则证明，但当前的血小板隔离程序需要血小板渗透和相对繁琐的纯化方法来进行最佳的确定，从而限制了更广泛的适用性。在这笔赠款任期期间，我们建议开发一个用于高通量血小板转录组分析的小型化系统。在特定的目标1中，我们将适应并发展机械剪切作为从全血中分离超纯血小板（1 mL）的有效方法，然后进行mRNA分离和代表性的转录放大，以独特的RNA产量为独特地适应。在特定的目标2中，我们将开发一个定制的血小板cDNA芯片，用于通过微阵列分析确定的放大血小板mRNA保真度的确认性研究。如果成功，该项目将开发适当的基础架构和方法，以更全面地分析较大的数据集，这是该领域调查领域的主要长期目标。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以及针对大型患者人群的分子诊断具有相当大的影响。