MicroRNAs (miRNAs) are a significant new class of biomolecules with potential to enhance human health. In the last three years, miRNA publications have increased exponentially as researchers have discovered the potential importance of these small RNAs in human cancer, neurological disease, and viral infection. Ambion researchers have worked to develop the tools necessary to understand the relationship between miRNAs and human health. Our results and the results from other institutions indicate that miRNAs may provide useful markers for the development of diagnostic and prognostic assays. In addition, there are hints that miRNAs might play an active role in disease progression, making them candidates for therapeutic intervention. The goal of this proposal is to develop rapid, sensitive, high-throughput methods to quantify the levels of small RNA molecules such as microRNAs (miRNA) in biological samples. These small RNAs have emerged as important regulators of gene expression which are critical for development and disease progression. Unfortunately, there currently are no rapid, quantitative methods available to measure levels of miRNA in clinical samples. The proposed research will result in the development of kits and reagents to rapidly quantify miRNAs in samples derived from tissues or cell culture. During Phase I, we invented a qRT-PCR based method to convert target miRNA molecules into cDNA and which are then amplified and detected using PCR. This method enables the specific detection of individual miRNAs in RNA samples. In Phase II we propose to optimize the method to facilitate its commercial use in research and diagnostic applications. Our Phase II research will improve sensitivity and specificity, permit simultaneous detection of multiple miRNAs in a single reaction, and enable detection on a Luminex liquid bead array platform. These improvements will enable rapid miRNA profiling for basic research, clinical validation of diagnostic and therapeutic miRNA candidates, and put a system in place for rapid development of miRNA-based clinical diagnostic assays. [unreadable] [unreadable] [unreadable]