Department of Bioengineering, California Institute of Technology, Pasadena, California, USA.
Nat Biotechnol. 2010 Nov;28(11):1208-12. doi: 10.1038/nbt.1692. Epub 2010 Oct 31.
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.
原位杂交方法可实现对完整生物样本中 mRNA 表达的定位。目前的方法难以在整个脊椎动物胚胎中同时定位多个靶标 mRNA，这是在最接近人类发育和疾病的系统中研究相互作用的调控元件时面临的重大限制。在这里，我们报告了一种基于杂交链反应（HCR）正交扩增的多路复用荧光原位杂交方法。在这种方法中，与 mRNA 靶标互补的 RNA 探针触发链反应，其中荧光标记的 RNA 发夹自组装成连接的荧光扩增聚合物。这些扩增级联的可编程性和序列特异性使多个 HCR 放大器能够在同一时间在同一样本中正交工作。在固定的全胚胎和切片斑马鱼胚胎中同时对 5 个靶标 mRNA 进行成像时，可实现稳健的性能。HCR 放大器具有深的样本穿透性、高的信号背景比和尖锐的信号定位。
