Amplifying mRNA Sequences from Small Amounts of Tissue

从少量组织中扩增 mRNA 序列

• 批准号: 7108072
• 负责人: PAUL D SIEBERT
• 金额: $ 14.33万
• 依托单位: SYSTEM BIOSCIENCES, LLC (SBI)
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-01 至 2007-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7108072
• 关键词: RNA; genes; tissues

DESCRIPTION (provided by applicant): Identifying differently expressed genes and their mRNA transcripts is a valuable starting point in understanding the molecular mechanisms underlying biological processes in normal and diseased states. Two widely used methods to facilitate these studies are Gene Expression Microarrays and Quantitative Reverse Transcriptase-linked Polymerase Chain Reaction. Although these are powerful techniques, they require microgram quantities of input RNA. In many cases, these amounts of RNA can be difficult or impossible to obtain. This is particularly the case when examining mRNA levels in primary cells, tissue biopsies, micro-dissected tissues and single cells. In these situations, a means to generate sufficient cDNA for multiple tests from small amounts of RNA for analysis would be very valuable. In the Phase I Research we propose to develop such a method, and utilize a set on non-random DNA primers for the synthesis and amplification of cDNA. This method will have advantages over current methods in terms of coverage of mRNA sequence representation, ability to work with partially degraded RNA, simplicity, time savings and cost. Proof of concept has been established and a commercialization plan is described.

描述（由申请人提供）：鉴定不同表达的基因及其mRNA转录物是理解正常和疾病状态下生物学过程的分子机制的有价值的起点。两种广泛使用的方法，以促进这些研究是基因表达微阵列和定量逆转录酶连接的聚合酶链反应。虽然这些都是强大的技术，但它们需要微克数量的输入RNA。在许多情况下，这些量的RNA可能很难或不可能获得。当检查原代细胞、组织活检、显微解剖组织和单细胞中的mRNA水平时，情况尤其如此。在这些情况下，从用于分析的少量RNA产生用于多次测试的足够cDNA的手段将是非常有价值的。在第一阶段的研究中，我们建议开发这样的方法，并利用一套非随机的DNA引物的cDNA的合成和扩增。该方法在mRNA序列表达的覆盖率、与部分降解的RNA一起工作的能力、简单性、节省时间和成本方面优于现有方法。概念验证已经确立，并描述了商业化计划。