Third-generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust

被引:591
作者
Choi, Harry M. T. [1 ]
Schwarzkopf, Maayan [1 ]
Fornace, Mark E. [2 ]
Acharya, Aneesh [1 ]
Artavanis, Georgios [1 ]
Stegmaier, Johannes [3 ,4 ,5 ]
Cunha, Alexandre [3 ,6 ]
Pierce, Niles A. [1 ,7 ,8 ]
机构
[1] CALTECH, Div Biol & Biol Engn, Pasadena, CA 91125 USA
[2] CALTECH, Div Chem & Chem Engn, Pasadena, CA 91125 USA
[3] CALTECH, Beckman Inst, Ctr Adv Methods Biol Image Anal, Pasadena, CA 91125 USA
[4] Karlsruhe Inst Technol, Inst Automat & Appl Informat, D-76344 Karlsruhe, Germany
[5] Rhein Westfal TH Aachen, Inst Imaging & Comp Vis, D-52074 Aachen, Germany
[6] CALTECH, Ctr Data Driven, Pasadena, CA 91125 USA
[7] CALTECH, Div Engn & Appl Sci, Pasadena, CA 91125 USA
[8] Univ Oxford, Weatherall Inst Mol Med, Oxford OX3 9DS, England
来源
DEVELOPMENT | 2018年 / 145卷 / 12期
基金
美国国家卫生研究院; 美国国家科学基金会;
关键词
Automatic background suppression; dHCR imaging; In situ HCR v3.0; Multiplexed quantitative in situ hybridization; qHCR flow cytometry; qHCR imaging; ROLLING-CIRCLE AMPLIFICATION; WHOLE-MOUNT; SIGNAL AMPLIFICATION; GENE-EXPRESSION; CHICK-EMBRYOS; DEPOSITION; RNAS;
D O I
10.1242/dev.165753
中图分类号
Q [生物科学];
学科分类号
07 ; 0710 ; 09 ;
摘要
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.
引用
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页数:10
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