Ligo-miR - A Multiplexed Single Molecule Ligation Assay for miRNA Profiling

Information

  • Research Project
  • 8550117
  • ApplicationId
    8550117
  • Core Project Number
    R43GM103360
  • Full Project Number
    5R43GM103360-02
  • Serial Number
    103360
  • FOA Number
    PA-10-122
  • Sub Project Id
  • Project Start Date
    9/24/2012 - 12 years ago
  • Project End Date
    4/30/2015 - 9 years ago
  • Program Officer Name
    MAAS, STEFAN
  • Budget Start Date
    9/1/2013 - 11 years ago
  • Budget End Date
    4/30/2015 - 9 years ago
  • Fiscal Year
    2013
  • Support Year
    02
  • Suffix
  • Award Notice Date
    7/15/2013 - 11 years ago
Organizations

Ligo-miR - A Multiplexed Single Molecule Ligation Assay for miRNA Profiling

DESCRIPTION (provided by applicant): Ligo-miR - A Multiplexed Single Molecule Ligation Assay for miRNA profiling MicroRNAs (miRNA) are short, noncoding RNAs with pervasive roles throughout gene expression in cellular processes such as differentiation and disease states such as cancer. Uncovering the roles of these molecules in development and tumorigenesis are key steps to the discovery of robust, new biomarkers and potential disease cures. The ability to profile miRNA expression at single cell resolution across a tumor mass could lead to targeted therapies that are effective at eliminating rather than merely shrinking tumors. No existing miRNA analysis method combines high sensitivity with true multiplexing and small volume capability. In this Phase I SHIFT proposal, a PCR-free, multiplex ligation assay for miRNA profiling called Ligo-miR will be developed. Hybridization and ligation of locked nucleic acid probes will be used to generate miRNA specific ligation products encoded by length. The ligation products will then be directly identified and quantified using microfluidic single molecul free solution hydrodynamic separation (SML-FSHS). The ligation mechanism will enable Ligo-miR to perform multiplex detection of up to 20 miRNA per reaction while the single molecule analysis platform will enable PCR-free detection with a sensitivity of <20 copies and sample volume <10 pL. This unique combination of high sensitivity and near-zero sample volume will form the foundation for a Phase II single cell miRNA profiling platform. Furthermore, this architecture can be easily scaled to even higher degrees of multiplexing (>50-plex) and throughput through microfluidics. In Aim 1, we will develop the fundamental Ligo-miR assay using synthetic RNA targets to mimic 3 classical miRNAs, let-7a, miR-16, and miR-21. In Aim 2, we will design a SML-FSHS microfluidic device to analyze the ligation products generated in Aim 1. In Aim 3, we will integrate these techniques into a multiplexed assay that can detect 20 miRNAs per reaction. Finally, in Aim 4, we will use the 20-plex assay to profile miRNA in 3 human cancer cell lines and 3 normal tissues. The Ligo-miR results will then be compared to published microarray and RT-PCR data. Such a method not only has applications in miRNA tumor profiling but also in other applications with rare samples such as clinical diagnostics using circulating tumor cells and cell-free miRNA.

IC Name
NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
  • Activity
    R43
  • Administering IC
    GM
  • Application Type
    5
  • Direct Cost Amount
  • Indirect Cost Amount
  • Total Cost
    199842
  • Sub Project Total Cost
  • ARRA Funded
    False
  • CFDA Code
    859
  • Ed Inst. Type
  • Funding ICs
    NIGMS:199842\
  • Funding Mechanism
    SBIR-STTR RPGs
  • Study Section
    ZRG1
  • Study Section Name
    Special Emphasis Panel
  • Organization Name
    CIRCULOMICS, INC.
  • Organization Department
  • Organization DUNS
    830377581
  • Organization City
    BALTIMORE
  • Organization State
    MD
  • Organization Country
    UNITED STATES
  • Organization Zip Code
    212112840
  • Organization District
    UNITED STATES