miR-SEQ: Highly efficient targeted quantification of extracellular miRNAs by Next Generation Sequencing

DESCRIPTION provided by applicant Recent discoveries highlight the importance of extracellular miRNAs ex miRNAs as promising noninvasive biomarkers for many diseases It has also been reported that several ex miRNAs species might be important for cell cell signaling While there is still controversy about their biological importance it is clear that ex miRNAs hold promise as biomarkers for different pathologies It has been reported that distinct expression patterns of miRNAs are associated with cancer and other diseases and ex miRNAs may reflect these signatures Biofluids such as plasma can be accessed with minimal invasiveness unlike tissue biopsies This together with the high stability of ex miRNAs in biofluids makes them attractive for use in molecular diagnostics compared to other more labile biomolecules Despite these advantages current techniques are inadequate for specific and reliable quantification of miRNAs in biofluids Microarrays and RT qPCR are currently the preferred tools for expression profiling of ex miRNAs although each has major drawbacks Microarrays suffer from low sensitivity low dynamic range and the inability to distinguish closel related miRNA sequences while RT qPCR has limited multiplexing capability and amplification biases While next generation sequencing NGS is superior in many of these respects its reliability for ex miRNA profiling in biofluids is limited due to bias underdetection towards particular miRNAs overwhelming amounts of unrelated sequencing data the need for gel purification of amplicons and its high cost Here we propose a new approach for constructing ex miRNA sequencing libraries that addresses these problems Called miR SEQ it incorporates new enzymatic steps to simplify and increase the sensitivity of ligation reactions and it involves a targeted sequencing approach that allows for the quantification of rare ex miRNAs that would otherwise be represented by low numbers of reads making their quantification problematic and or expensive Although our method is applicable to any miRNA to establish proof of concept this Phase I proposal focuses on the ex miRNAs reported to be most abundant in plasma Once we achieve reliable quantification for known plasma miRNAs we expect to use the method to discover and validate ex miRNAs biomarkers in human plasma for Parkinsonandapos s Disease and develop miR SEQ diagnostic assays using those validated biomarkers PUBLIC HEALTH RELEVANCE The goal of this grant application is to solve a detection problem in profiling microRNAs miRNAs from biofluids using next generation sequencing NGS methods Current methods lead to serious undercounting of many miRNAs due to bias in the enzymatic reactions used to prepare the samples for NGS and because of their intrinsically low concentrations in biofluids Eliminating this bias will help to fully realize the potential of miRNAs as biomarkers of cancer and other diseases In addition this method may help reduce the cost of sequencing clinical samples making this powerful diagnostic tool more accessible to patients