Method for Analyzing Non-poly(A) RNA Transcripts

分析非多聚 (A) RNA 转录物的方法

• 批准号: 7485544
• 负责人: JONATHAN MEADE
• 金额: $ 14.98万
• 依托单位: GENHUNTER CORPORATION
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7485544
• 关键词: Bypass; Cells; Classification; Complementary DNA; DNA Microarray Chip; DNA Microarray format; Detection; Eukaryotic Cell; Gene Expression; Genetic Transcription; In Vitro; Mammalian Cell; Messenger RNA; Methods; Microarray Analysis; Pattern; Phase; Poly A; Poly(A) Tail; Poly(A)+ RNA; Population; RNA; Reaction; Ribosomal RNA; Small Business Technology Transfer Research; Testing; Transcript; Transfer RNA; mRNA Differential Displays; mRNA Expression

DESCRIPTION (provided by applicant): Much efforts in gene expression analysis in the past have been focused mainly on the messenger RNAs (mRNAs), thanks to the availability of Differential Display (DD), SAGE and DNA microarray technologies, which all target the poly(A) tails present in most eukaryotic mRNAs. The recent discovery of a large microRNA population begged the question of whether there exists additional yet to be discovered RNAs species in a eukaryotic cell besides mRNA, rRNA and tRNA. However, in contrast to the analysis of mRNA expression, analogous methods for an accurate, comprehensive detection and analysis of any nonpolyadenylated RNA have been lacking. Here we describe a systematic approach for the detection and identification of any non-polyadenylated RNAs in a eukaryotic cell. The method involves first in vitro enzymatic addition of a poly(A) tail to all non-poly(A) RNAs in a cell followed by fluorescent Differential Display (FDD) comparison of cDNA patterns before and after poly(A) addition. With the proof of principle established for the method, two well defined specific aims are formulated in this Phase I STTR application to further optimize and streamline the method for a more accurate and comprehensive screen for nonpolyadenylated RNA species expression in any eukaryotic cell. Specific Aim 1: Systematic Analysis of Non-poly(A) RNA Expression in Eukaryotic Cells by Differential Display a: Optimization of poly(A) tailing reaction of NPA-DD b: Poly(A) tailing of total RNA following the depletion of poly(A) RNA and comparison of NPA-DD with tiling arrays, c: NPA-DD bypassing ribosomal RNA detection. Specific Aim 2: Comprehensive Test Screens for Non-poly(A) RNA Expression in Mammalian Cells.

描述（由申请人提供）：由于差分显示（DD）， SAGE和DNA微阵列技术的可用性，过去基因表达分析的大部分工作主要集中在信使rna （mrna）上，这些技术都针对大多数真核mrna中存在的聚(A)尾。最近大量microRNA种群的发现提出了一个问题，即真核细胞中除了mRNA、rRNA和tRNA外，是否还存在其他尚未发现的rna物种。然而，与mRNA表达的分析相反，缺乏准确、全面检测和分析任何非聚腺苷化RNA的类似方法。在这里，我们描述了一种系统的方法来检测和鉴定真核细胞中任何非聚腺苷化的rna。该方法包括首先在体外酶促下将poly(a)尾部添加到细胞中所有非poly(a) rna上，然后通过荧光差异显示（FDD）比较添加poly(a)前后的cDNA模式。随着该方法的原理证明的建立，在本I期STTR申请中制定了两个明确的具体目标，以进一步优化和简化该方法，以便更准确和全面地筛选任何真核细胞中非聚腺苷化RNA物种的表达。具体目标1：用差异显示法系统分析真核细胞中非聚(A) RNA的表达A：优化NPA-DD的聚(A)尾化反应b：聚(A) RNA耗散后总RNA的聚(A)尾化以及NPA-DD与平铺阵列的比较c: NPA-DD绕过核糖体RNA检测。特异性目标2：哺乳动物细胞中非聚(A) RNA表达的综合测试筛选。