Combinatorial analysis of mRNA expression patterns in mouse embryos using hybridization chain reaction

Cold Spring Harbor Protocols
David HussRusty Lansford

Abstract

Multiplexed fluorescent hybridization chain reaction (HCR) and advanced imaging techniques can be used to evaluate combinatorial gene expression patterns in whole mouse embryos with unprecedented spatial resolution. Using HCR, DNA probes complementary to mRNA targets trigger chain reactions in which metastable fluorophore-labeled DNA HCR hairpins self-assemble into tethered fluorescent amplification polymers. Each target mRNA is detected by a probe set containing one or more DNA probes, with each probe carrying two HCR initiators. For multiplexed experiments, probe sets for different target mRNAs carry orthogonal initiators that trigger orthogonal DNA HCR amplification cascades labeled by spectrally distinct fluorophores. As a result, in situ amplification is performed for all targets simultaneously, and the duration of the experiment is independent of the number of target mRNAs. We have used multiplexed fluorescent in situ HCR and advanced imaging technologies to address questions of cell heterogeneity and tissue complexity in craniofacial patterning and anterior neural development. In the sample protocol presented here, we detect three different mRNA targets: Tg(egfp), encoding the enhanced green fluorescent protein (GFP) tra...Continue Reading

References

Oct 20, 2004·Proceedings of the National Academy of Sciences of the United States of America·Robert M Dirks, Niles A Pierce
Nov 3, 2010·Nature Biotechnology·Harry M T ChoiNiles A Pierce

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Citations

Mar 4, 2017·The Journal of Physical Chemistry. B·Xiaoping OlsonWilliam L Hughes
Oct 6, 2016·Development·Harry M T ChoiNiles A Pierce
Apr 16, 2019·Frontiers in Cell and Developmental Biology·David J HussRusty Lansford
Feb 19, 2020·Nature Neuroscience·Caitlin M RodriguezPeter K Todd
Jan 9, 2021·PloS One·Adam Joseph Ronald PondBenjamin Steventon
May 8, 2018·ACS Sensors·Erik E AugspurgerMehmet V Yigit

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