Journal
NATURE BIOTECHNOLOGY
Volume 28, Issue 11, Pages 1208-U103Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nbt.1692
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Funding
- US National Institutes of Health [R01 EB006192, P50 HG004071]
- National Science Foundation [CCF-0448835, CCF-0832824]
- Beckman Institute at Caltech
- Division of Computing and Communication Foundations
- Direct For Computer & Info Scie & Enginr [832824] Funding Source: National Science Foundation
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In situ hybridization methods enable the mapping of mRNA expression within intact biological samples(1,2). With current approaches, it is challenging to simultaneously map multiple target mRNAs within whole-mount vertebrate embryos(3-6), representing a significant limitation in attempting to study interacting regulatory elements in systems most relevant to human development and disease. Here, we report a multiplexed fluorescent in situ hybridization method based on orthogonal amplification with hybridization chain reactions (HCR)(7). With this approach, RNA probes complementary to mRNA targets trigger chain reactions in which fluorophore-labeled RNA hairpins self-assemble into tethered fluorescent amplification polymers. The programmability and sequence specificity of these amplification cascades enable multiple HCR amplifiers to operate orthogonally at the same time in the same sample. Robust performance is achieved when imaging five target mRNAs simultaneously in fixed whole-mount and sectioned zebrafish embryos. HCR amplifiers exhibit deep sample penetration, high signal-to-background ratios and sharp signal localization.
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