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Published October 31, 2010 | Supplemental Material + Accepted Version
Journal Article Open

Programmable in situ amplification for multiplexed imaging of mRNA expression


In situ hybridization methods enable the mapping of mRNA expression within intact biological samples. With current approaches, it is challenging to simultaneously map multiple target mRNAs within whole-mount vertebrate embryos, 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). 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.

Additional Information

© 2010 Nature Publishing Group. Received 28 June; accepted 24 September; published online 31 October 2010. We thank V.A. Beck, J.S. Bois, S. Venkataraman, J.R. Vieregg and P. Yin for discussions. We thank C. Johnson and A.J. Ewald for performing preliminary studies. We thank J.N. Zadeh for the use of unpublished software. We thank the Caltech Biological Imaging Center and A. Collazo of the House Ear Institute for the use of multispectral confocal microscopes. This work was funded by the US National Institutes of Health (R01 EB006192 and P50 HG004071), the National Science Foundation (CCF-0448835 and CCF-0832824) and the Beckman Institute at Caltech. Contributions: S.E.F. and N.A.P. conceived the application of HCR to multiplexed bioimaging; H.M.T.C., J.Y.C., J.E.P. and N.A.P. engineered HCR hairpins for use in stringent hybridization buffers; H.M.T.C. and N.A.P. designed the experiments; H.M.T.C. performed the experiments; L.A.T. selected targets, provided technical guidance and performed the control experiments using traditional in situ hybridization; H.M.T.C., L.A.T., S.E.F. and N.A.P. analyzed the data; H.M.T.C. and N.A.P. wrote the manuscript; and all authors edited the manuscript. The authors declare competing financial interests in the form of US patents and pending US and EU patents.

Attached Files

Accepted Version - nihms240064.pdf

Supplemental Material - nbt.1692-S1.pdf

Supplemental Material - nbt.1692-S2.avi

Supplemental Material - nbt.1692-S3.avi


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August 22, 2023
October 17, 2023