Division of Biology & Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
Division of Chemistry & Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
Development. 2018 Jun 26;145(12):dev165753. doi: 10.1242/dev.165753.
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 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. 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.
基于杂交链式反应（HCR）机制的杂交技术解决了阻碍多种生物体中 mRNA 表达成像的数十年难题，提供了独特的多重检测、定量、灵敏度、分辨率和多功能性的结合。在这里，我们使用第三代 HCR 的探针和扩增子来增强这些功能，这些探针和扩增子结合在一起，在整个方案中提供自动背景抑制，确保试剂即使在样本中非特异性结合也不会产生扩增背景。自动背景抑制极大地提高了性能和稳健性，将更高的信号与背景比的优势与使用未经优化的探针组进行新靶标和新生物体分析的便利性相结合。HCR v3.0 支持三种多重定量分析模式：（1）qHCR 成像-在整个脊椎动物胚胎的解剖学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分析；（3）dHCR 成像-通过厚的自体荧光样本中的单分子成像进行数字 mRNA 绝对定量。
