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. ? ? ?
