Programmable in situ amplification for multiplexed imaging of mRNA expression

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Abstract
The simultaneous detection of multiple mRNA species in thick tissues or whole-mount embryos has remained technically challenging. Choi et al. present a method based on the triggered polymerization of RNA stem-loop structures that allows the distribution of up to five mRNAs in intact zebrafish embryos to be imaged at the same time. In situ hybridization methods enable the mapping of mRNA expression within intact biological samples1,2. With current approaches, it is challenging to simultaneously map multiple target mRNAs within whole-mount vertebrate embryos3,4,5,6, 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.