The aim of this SBIR application is to develop a bead-based array system for the specific detection and classification of microRNAs (miRNAs). The discovery of miRNAs represents a paradigm shift that suggests the existence of many unknown cellular function and regulation mechanisms. Already, miRNAs have been found implicated in different cancers and leukemia. However, the existing methods for studying the expression of miRNAs are labor intensive, time consuming, and also lack specificity and sensitivity. A more efficient and accurate research and development tool is much in demand. We have developed a bead-based array method that integrates the xMAP technology platform with the locked nucleic acid (LNA) technology. The method, called xMAP-MP for xMAP-based miRNA profiling, uses beads coupled to a capture oligonucleotide (oligo) and a biotin labeled detecting oligo to quantitatively detect and classify miRNA. With this method, multiple miRNAs can be studied together in one reaction. Preliminary studies have shown that the assay is highly specific and sensitive: miRNAs can be detected using only 100 ng of total RNA. This is more than 100 times more sensitive than the traditional Northern blot method. The xMAP-MP is also very efficient: samples do not need to be labeled; hybridization takes only 30 minutes; and detection and data acquisition takes only a minute. The entire procedure can be finished within an hour. Furthermore, the multiplex capability of the xMAP technology platform allows the study of up to 100 miRNAs in one assay. The proposed study has four specific aims: 1) Select 20 cancer related miRNA targets and design a multiplexed detection system for these miRNAs; 2) Design internal and external controls for the assay system; 3) Develop and optimize a standard assay protocol, and 4) Use the prototype assay to study cancer samples.
