Combinatorial analysis of mRNA expression patterns in mouse embryos using hybridization chain reaction.
Cold Spring Harb Protoc
; 2015(3): 259-68, 2015 Mar 02.
Article
en En
| MEDLINE
| ID: mdl-25734068
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) transgene (typically used as a control); Twist1, encoding a transcription factor involved in cell lineage determination and differentiation; and Pax2, encoding a transcription factor expressed in the mid-hindbrain region of the mouse embryo.
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
ARN Mensajero
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Regulación del Desarrollo de la Expresión Génica
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Perfilación de la Expresión Génica
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Técnicas de Amplificación de Ácido Nucleico
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Embrión de Mamíferos
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Hibridación de Ácido Nucleico
Límite:
Animals
Idioma:
En
Revista:
Cold Spring Harb Protoc
Año:
2015
Tipo del documento:
Article