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    • 8. 发明公开
    • SINGLE MOLECULE RNA DETECTION
    • EP3198037A4
    • 2018-03-21
    • EP15843589
    • 2015-09-18
    • UNIV CALIFORNIA
    • ZHONG SHENGBIASE FERNANDO
    • C12Q1/6841C12Q1/682
    • C12Q1/6841C12Q1/682C12Q1/6825C12Q2537/143C12Q2563/143C12Q2543/10C12Q2563/107C12Q2563/149
    • Quantification of RNAs is one of the most essential tools to characterize cells. This tool is widely used in disease diagnosis, pharmacogenomics, and drug development. Single cell RNA fluorescent in situ hybridization (smRNA-FISH) revolutionized RNA detection and quantification by detecting every single RNA molecule of a gene. However, this technology is incapable of assaying relatively short RNAs, and it suffers from high cost and low throughput. Here, we describe a technology that simultaneously overcomes the three drawbacks using conventional instrumentation. This QD-smRNA-FISH technology uses hybridization of quantum dot-labeled DNA oligonucleotides to the RNA molecules for visualization and counting. Quantum dots (QDs) have been assumed inapplicable to counting individual RNA molecules, due to the well-known blinking problem (display intermittency) (Medintz I L, Uyeda H T, Goldman E R, Mattoussi H (2005) Quantum dot bioconjugates for imaging, labelling and sensing. Nat Mater 4: 435-446). This problem has been circumvented by this new experimental design. In some embodiments described herein, the methods assemble several QDs to every target RNA molecule and leverage the complementation of the QDs to achieve an overall non-intermittent signal on each target molecule. We validated QD-smRNA-FISH by comparing its signals with those of standard smRNA-FISH. We successfully applied QD-smRNA-FISH to test the interaction of two RNAs, a task that cannot be accomplished with standard smRNA-FISH. The QD-smRNA-FISH method offers a highly accurate method for single RNA molecule detection and counting under standard fluorescent microscopes, and enables analysis of relatively short RNAs (