MicroRNAs (miRNAs) are a group of small, non-coding RNAs, approximately 19-23 nucleotides in length, that regulate gene expression by binding to sequences within the 3' untranslated regions of specific target mRNAs; a single miRNA might bind to and regulate as many as 200 diverse targets. Only recently discovered, and thought to be essential for cell proliferation and differentiation, it is clear that abnormalities in miRNA activity contribute to the pathogenesis of human diseases including cancer. Microarrays are ideal for high-throughput analysis of gene expression; however, when analyzing miRNA expression levels this is made difficult by their inherent small size (which provides little sequence for designing microarray capture sequences or attaching label) and the fact that they represent only ~0.01% of the mass of a total RNA sample. Typically, for microarray applications, fluorescent dyes such as Cy(tm)3 and Cy(tm)5 are added to miRNAs using a poly(A) polymerase-based method. Label IT(r) reagents (Mirus Bio) utilize alkylating chemistry to covalently link a detectable marker such as Cy3 and Cy5 directly to nucleic acids. Labeling miRNA samples with the Label IT reagents generates microarray expression profiles that consistently identify miRNAs that the enzymatic labeling methods fail to detect, including potential diagnostic markers documented in various cancers. Expression profiling of miRNAs for the diagnosis, treatment, and prevention of human diseases is a rapidly expanding field of research that is critically dependent on the successful identification of all miRNA species present in the sample. To determine the number of discrepancies in the miRNA expression profiles generated using Label IT technology and enzymatic labeling methodologies in microarray analyses, miRNA-enriched samples from human tumor/normal cell line pairs will be labeled using chemical and enzymatic methods, hybridized to multiple replicates of at least two different commercial microarrays, and the differences in expression profiles validated by independent methods. To optimize Label IT labeling for clinically relevant samples (where the amount of miRNA may be limiting), a systematic examination of various miRNA enrichment and amplification procedures will be performed using one tumor/normal cell line pair, and will include a determination of the minimum amount of non-amplified material needed to generate representative microarray expression profiles and the development of universal Label IT labeling protocols for processing clinical RNA samples. Furthermore, the Label IT technology will be optimized for microarray miRNA expression profiling of mouse and human cancer specimens. We anticipate that chemical labeling of miRNA from clinical research and diagnostic specimens will offer many critical advantages for accurate expression profiling compared to currently available enzymatic labeling methods. 7. Project Narrative MicroRNAs (miRNAs) are a group of small molecules that normally regulate cell growth, but they may cause diseases such as cancer when their expression is altered. Microarrays are a method for looking at the total expression of all genes in a given sample, and these expression profiles of miRNAs in diseased tissues may be used for diagnosis and to suggest treatment options. In contrast to other methods that use enzymes, Label IT(r) reagents chemically label miRNAs for examination by microarrays, resulting in a more accurate and complete expression profile compared to other labeling technologies with clear advantages for clinical research and diagnosis of human diseases. ? ? ?