Method for Analyzing Non-poly(A) RNA Transcripts

描述（由申请人提供）：由于差异显示（DD）、SAGE和DNA微阵列技术的可用性，过去基因表达分析中的许多努力主要集中在信使RNA（mRNA）上，这些技术都靶向大多数真核mRNA中存在的poly（A）尾。最近发现的大量microRNA群体提出了一个问题，即除了mRNA，rRNA和tRNA之外，真核细胞中是否存在其他尚未发现的RNA种类。然而，与mRNA表达的分析相反，缺乏用于准确、全面检测和分析任何非多聚腺苷酸化RNA的类似方法。在这里，我们描述了一个系统的方法，用于检测和鉴定任何非多聚腺苷酸化的RNA在真核细胞。该方法包括首先在体外酶促添加poly（A）尾至细胞中的所有非poly（A）RNA，然后在添加poly（A）之前和之后荧光差异显示（FDD）比较cDNA模式。在为该方法建立的原理证明的基础上，在该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，从而更准确和全面地筛选任何真核细胞中的非聚腺苷酸化RNA种类表达。具体目标1：通过差异显示系统分析真核细胞中非多聚（A）RNA表达a：NPA-DD的多聚（A）加尾反应的优化B：多聚（A）RNA耗尽后总RNA的多聚（A）加尾和NPA-DD与平铺阵列的比较，c：NPA-DD绕过核糖体RNA检测。具体目标2：在哺乳动物细胞中进行非poly（A）RNA表达的全面测试筛选。

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.