Third-generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust
Third-generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust
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DOI：
10.1242/dev.165753
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发表时间：
2018-06-01
期刊：
影响因子：
4.6
通讯作者：
Pierce, Niles A.
中科院分区：
文献类型：
--
作者：
Choi, Harry M. T.;Schwarzkopf, Maayan;Pierce, Niles A.
基于杂交链式反应 (HCR) 机制的原位杂交解决了数十年来阻碍多种生物体 mRNA 表达成像的挑战，提供了多重、定量、灵敏度、分辨率和多功能性的独特组合。在这里，借助第三代原位 HCR，我们使用探针和放大器增强了这些功能，它们结合起来在整个协议中提供自动背景抑制，确保试剂即使在样品内非特异性结合也不会产生放大的背景。自动背景抑制显着提高了性能和鲁棒性，将较高信号背景比的优点与使用未优化的探针组用于新目标和生物体的便利性相结合。原位 HCR v3.0 支持三种多重定量分析模式：(1) qHCR 成像 - 在整体脊椎动物胚胎的解剖背景下进行亚细胞分辨率的模拟 mRNA 相对定量； (2) qHCR流式细胞术——模拟mRNA相对定量，用于哺乳动物和细菌细胞的高通量表达谱分析； (3) dHCR 成像 - 通过厚的自发荧光样品中的单分子成像进行数字 mRNA 绝对定量。
In situ hybridization based on the mechanism of the hybridization chain reaction (HCR) has addressed multi-decade challenges that impeded imaging of mRNA expression in diverse organisms, offering a unique combination of multiplexing, quantitation, sensitivity, resolution and versatility. Here, with third-generation in situ HCR, we augment these capabilities using probes and amplifiers that combine to provide automatic background suppression throughout the protocol, ensuring that reagents will not generate amplified background even if they bind non-specifically within the sample. Automatic background suppression dramatically enhances performance and robustness, combining the benefits of a higher signal-to-background ratio with the convenience of using unoptimized probe sets for new targets and organisms. In situ HCR v3.0 enables three multiplexed quantitative analysis modes: (1) qHCR imaging - analog mRNA relative quantitation with subcellular resolution in the anatomical context of whole-mount vertebrate embryos; (2) qHCR flow cytometry - analog mRNA relative quantitation for high-throughput expression profiling of mammalian and bacterial cells; and (3) dHCR imaging - digital mRNA absolute quantitation via single-molecule imaging in thick autofluorescent samples.