Ligo-miR - A Multiplex Assay Platform for Targeted miRNA Profiling

DESCRIPTION provided by applicant MiRNA have the potential to revolutionize both cancer detection and therapeutics as they exert control over nearly every cellular process from differentiation to growth and senescence Although over miRNA are predicted to exist studies have suggested that smaller subsets of only miRNA are often the critical players in any specific disease or cellular process However existing microarray and qRT PCR techniques are too slow and too expensive to screen large numbers of samples across these targeted miRNA panels a critical task in the validation of miRNA cancer tests In Phase I we developed a multiplexed ligation assay to analyze miRNA in a single reaction called Ligo miR We demonstrated that Ligo miR expression profiles had high correlation to the gold standard Taqman qRT PCR assays R Yet Ligo miR required x less hands on time had x less cost and had x higher overall sample throughput successfully achieving the goal of fast and low cost targeted miRNA profiling Ligo miR incorporates sequential ligation steps to generate miRNA specific reaction products encoded by length This flexible length based multiplexing is specifically designed to enable detection using an array of DNA sizing methods including polyacrylamide gel electrophoresis PAGE capillary electrophoresis CE and single molecule free solution hydrodynamic separation SML FSHS such that sensitivity multiplex capability and sample throughput can be tailored a wide spectrum of miRNA applications and user requirements In this Phase II SBIR proposal we will leverage the Phase I results to develop a platform of commercial products based on Ligo miR technology for broad research applications First Ligo miR EZ will be developed for general profiling of cells and tissues using fluorescent PAGE detection to achieve a sensitivity of copies per cell Nearly all molecular labs will have a thermal cycler and PAGE apparatus making fast and low cost multiplex analysis available to all Next Ligo miR CE will be developed for applications requiring the highest sensitivity and throughput such as single cell analysis or clinical analysis of blood It incorporates automated CE detection to increase sample throughput to thousands of samples per day and a post amplification to boost sensitivity to single cell levels Then Ligo miR HD will be developed for challenging applications using limited samples or that could benefit from lab on a chip integration such as FFPE analysis and single cell It uses microfluidic single molecule technology for high sensitivity detection using minute amounts of sample Finally we will perform a technical validation and benchmark each Ligo miR assay against Taqman qRT PCR PUBLIC HEALTH RELEVANCE Targeted panels of miRNA can be used as biomarkers with remarkable specificity and unprecedented scope to detect and manage cancer However existing methods are too expensive and too slow to perform large scale validation studies necessary for clinical translation This project will create new assay platform for fast and low cost targeted miRNA profiling that enable large scale studies at a fraction of the cost and time of existing techniques to speed the clinical translation of these promising cancer tests