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.
Division of Biology & Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
Maayan
M
Schwarzkopf
Division of Biology & Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA Division of Engineering & Applied Science, California Institute of Technology, Pasadena, CA 91125, USA niles@caltech.edu.
Niles A
NA
Pierce
D015152:Blotting, Northern / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis / Q000737:chemistry; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D035683:MicroRNAs / Q000032:analysis* / Q000235:genetics*; D015347:RNA Probes / Q000032:analysis / Q000737:chemistry; D013997:Time Factors
D005456:Fluorescent Dyes; D035683:MicroRNAs; D015347:RNA Probes