Expression Profiling of microRNAs with Bead Array

DESCRIPTION (provided by applicant): The aim of this SBIR application is to develop a bead based array system for the specific detection and classification of microRNAs. The discovery of miRNAs representing a paradigm shift that suggested the existence of many unknown cellular function and regulation mechanisms. Already, miRNAs have been found implicated in different cancers and leukemia. MicroRNA has all the characteristics to become new class of biomarker for pharmaceutical applications. However, the existing methods for studying the expression of miRNA are labor intensive, time consuming, and also lacks the specificity and sensitivity. A more efficient and accurate research and development tool is much in demand. We have developed a bead array based method that integrated the xMPA technology platform with the LNA (locked nucleic acid) technology. The method, called xMAP-MP for xMAP based miRNA profiling, used beads coupled capture 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 100ng of total RNA. It detects mature miRNAs preferentially over precursor miRNAs. It could also provide more accurate quantitative measurement of miRNA levels. Phase I study allowed us identified unique miRNA distributions in 10 normal tissues as well as in 10 solid tumor tissues. A "signature" expression profile was identified for breast cancer. 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 three specific aims: (1)Further develop and increase the coverage of detectable microRNAs. The current panels can detect 115 microRNA targets. We would like to expand the panels so that they can detect approximately 250 known microRNAs. (2)Use clinical samples and cancer cell lines to develop and evaluate "signature" panels for four malignancies, including breast cancer, prostate cancer, lymphoma, and glioma. (3) Prepare for commercialization by developing assay-specific software, and establishing GMP manufacture and quality control protocols. MicroRNA has all the characteristics to become the new class of biomarker for pharmaceutical applications. However, the existing methods for studying the expression of miRNA are labor intensive, time consuming, and also lacks the specificity and sensitivity. A more efficient and accurate research and development tool is much in demand. We have developed a bead array based method which allows multiple miRNAs to be studied together in one reaction.