Cellular microRNAs (miRNAs) are a newly discovered class of single-stranded RNA molecules that are expressed in cells of plants and animals. There are an estimated 500-700 miRNA genes for which about 260 have been identified for each species so far. miRNAS have been demonstrated to be negative regulators of gene-expression via translation inhibition and mRNA degradation. Precise function for each miRNAs is currently the subject of intense research effort. miRNA expression is highly regulated and coordinated, different cell types under different physiological conditions appear to have a unique miRNA expression pattern. Increasing evidence suggests miRNA expression pattern could be indicative not only of the physiological state but also the disease state of cell or tissue such as cancer. Highly sensitive tools are needed for detection and expression profiling of miRNAs. Northern blot is the gold-standard for detection and quantification of miRNAs. Northern blot is a comparatively tedious process with low sensitivity. Methods for high throughput and sensitivity are needed. Mature miRNAs are from 17-24 nucleotides long; because their mall size and single stranded nature, efficient and faithful amplification by conventional polymerase chain reaction (PCR) is difficult to achieve. Most current methods for detection and profiling rely on the amplification of signals; the sensitivity of those methods is still far below the level can be achieved with PCR amplification. In this project, we propose to develop a novel multiplex PCR based miRNA amplification system. The amplification system is designed to faithfully amplify all miRNAs simultaneously without bias. Commercial applications for this amplification system are numerous, such as for quantitation of miRNA by real-time PCR, for highly sensitive detection required for diagnostic purpose, and for miRNA expression profiling. The Phase I research will validate the process; Phase II will further develop commercial applications for the novel miRNA amplification system. Project Narrative: This project is to develop a novel technology for the research and diagnostic purposes. The developed technology will provide essential tools for biomedical research and for disease diagnostics,? such as cancer, for which will definitely have a great impact in public health in general. ? ? ?
