Abstract: The present invention provides a PCR-free, multiplexed ligation assay for miRNA expression analysis that produces highly quantitative, 10-100 plex miRNA profiling in a single reaction. The inventive methods use a 2-step ligation assay to generate an array of miRNA specific ligation products that can be decoded and quantified by a size discrimination method such as gel electrophoresis or single molecule separation. One embodiment is a low-cost assay that can be performed using standard tools available in nearly all molecular biology laboratories. This assay requires nothing more than a gel apparatus and reader for detection. Other embodiments include use of magnetic beads and other size exclusion apparatus which give increasingly higher sensitivity, lower sample consumption, and reduced processing steps.

Abstract: The present invention provides a PCR-free, multiplexed ligation assay for miRNA expression analysis that produces highly quantitative, 10-100 plex miRNA profiling in a single reaction. The inventive methods use a 2-step ligation assay to generate an array of miRNA specific ligation products that can be decoded and quantified by a size discrimination method such as gel electrophoresis or single molecule separation. One embodiment is a low-cost assay that can be performed using standard tools available in nearly all molecular biology laboratories. This assay requires nothing more than a gel apparatus and reader for detection. Other embodiments include use of magnetic beads and other size exclusion apparatus which give increasingly higher sensitivity, lower sample consumption, and reduced processing steps.