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Published September 6, 2016 | Supplemental Material + Published
Journal Article Open

Multiplexed miRNA northern blots via hybridization chain reaction


Northern blots enable detection of a target RNA of interest in a biological sample using standard benchtop equipment. miRNAs are the most challenging targets as they must be detected with a single short nucleic acid probe. With existing approaches, it is cumbersome to perform multiplexed blots in which several RNAs are detected simultaneously, impeding the study of interacting regulatory elements. Here, we address this shortcoming by demonstrating multiplexed northern blotting based on the mechanism of hybridization chain reaction (HCR). With this approach, nucleic acid probes complementary to RNA targets trigger chain reactions in which fluorophore-labeled DNA hairpins self-assemble into tethered fluorescent amplification polymers. The programmability of HCR allows multiple amplifiers to operate simultaneously and independently within a blot, enabling straightforward multiplexing. We demonstrate simultaneous detection of three endogenous miRNAs in total RNA extracted from 293T and HeLa cells. For a given target, HCR signal scales linearly with target abundance, enabling relative and absolute quantitation. Using non-radioactive HCR, sensitive and selective miRNA detection is achieved using 2′OMe-RNA probes. The HCR northern blot protocol takes ∼1.5 days independent of the number of target RNAs.

Additional Information

© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. Received October 17, 2015; Revised May 4, 2016; Accepted May 24, 2016. We thank H.M.T. Choi, J.B. Sternberg, L.M. Hochrein and J.C. Peters for helpful discussions. National Institutes of Health [5R01EB006192]; National Science Foundation via the Molecular Programming Project [NSF-CCF-1317694]; Gordon and Betty Moore Foundation [GBMF2809]; Beckman Institute at Caltech; Guggenheim Fellowship (to N.A.P.); St Catherine's College, University of Oxford Christensen Fellowship (to N.A.P.). Funding for open access charge: National Institutes of Health [5R01EB006192]. Conflict of interest statement. The authors declare competing financial interests in the form of US and EPO patents and pending patents.

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