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Methods. 2013 Sep 15;63(2):188-99. doi: 10.1016/j.ymeth.2013.05.028. Epub 2013 Jun 29.

Dissecting non-coding RNA mechanisms in cellulo by Single-molecule High-Resolution Localization and Counting.

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Single Molecule Analysis in Real-Time (SMART) Center, University of Michigan, Ann Arbor, MI 48109-1055, USA.


Non-coding RNAs (ncRNAs) recently were discovered to outnumber their protein-coding counterparts, yet their diverse functions are still poorly understood. Here we report on a method for the intracellular Single-molecule High-Resolution Localization and Counting (iSHiRLoC) of microRNAs (miRNAs), a conserved, ubiquitous class of regulatory ncRNAs that controls the expression of over 60% of all mammalian protein coding genes post-transcriptionally, by a mechanism shrouded by seemingly contradictory observations. We present protocols to execute single particle tracking (SPT) and single-molecule counting of functional microinjected, fluorophore-labeled miRNAs and thereby extract diffusion coefficients and molecular stoichiometries of micro-ribonucleoprotein (miRNP) complexes from living and fixed cells, respectively. This probing of miRNAs at the single molecule level sheds new light on the intracellular assembly/disassembly of miRNPs, thus beginning to unravel the dynamic nature of this important gene regulatory pathway and facilitating the development of a parsimonious model for their obscured mechanism of action.


Non-coding RNA; RNA silencing; Single molecule microscopy; Single particle tracking; microRNA

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