Combinatorial analysis of mRNA expression patterns in mouse embryos using hybridization chain reaction.
Combinatorial analysis of mRNA expression patterns in mouse embryos using hybridization chain reaction.
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DOI：
10.1101/pdb.prot083832
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发表时间：
2015-03-02
影响因子：
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通讯作者：
Lansford, Rusty
中科院分区：
文献类型：
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作者：
Huss, David;Choi, Harry M T;Lansford, Rusty
多重荧光杂交链反应（HCR）和先进的成像技术可以用来评估组合基因表达模式在整个小鼠胚胎前所未有的空间分辨率。使用HCR，与mRNA靶互补的DNA探针触发链式反应，其中亚稳态荧光团标记的DNA HCR发夹自组装成系留的荧光扩增聚合物。通过含有一个或多个DNA探针的探针组检测每个靶mRNA，每个探针携带两个HCR起始子。对于多重实验，不同靶mRNA的探针组携带正交引发剂，其触发由光谱上不同的荧光团标记的正交DNA HCR扩增级联。因此，对所有靶点同时进行原位扩增，并且实验持续时间与靶mRNA的数量无关。我们已经使用多路荧光原位HCR和先进的成像技术，以解决颅面图案和前神经发育的细胞异质性和组织复杂性的问题。在这里提出的样本协议中，我们检测三种不同的mRNA靶：Tg（egfp），编码增强型绿色荧光蛋白（GFP）转基因（通常用作对照）; Twist 1，编码参与细胞谱系确定和分化的转录因子;和Pax 2，编码在小鼠胚胎中后脑区域表达的转录因子。
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.