Highly Sensitive and Multiplexed In Situ RNA Profiling with Cleavable Fluorescent Tyramide.
Highly Sensitive and Multiplexed In Situ RNA Profiling with Cleavable Fluorescent Tyramide.
复制标题
高度敏感和多路复用的原位RNA分析，可裂解荧光酪氨酰胺。
作者：
Xiao L;Labaer J;Guo J
要理解复杂生物系统的组成、调控和功能，就需要能够量化其原生细胞位置的多种转录本的工具。然而，目前的多路RNA成像技术受其相对较低的灵敏度或特异性的限制，这阻碍了其在研究高自荧光组织中的应用，如福尔马林固定石蜡包埋（FFPE）组织。为了解决这个问题，我们开发了一种具有高灵敏度和特异性的多路原位RNA分析方法。在这种方法中，转录本首先被靶向特异性寡核苷酸探针成对杂交。只有当这两个独立的探针串联杂交到靶标上时，才会发生随后的寡核苷酸杂交信号扩增。随后，利用辣根过氧化物酶（HRP）进一步放大信号，并用可切割荧光酪酰胺（CFT）对靶标进行染色。成像后，荧光团被化学切割，杂交探针被dna酶和甲酰胺剥离。通过RNA染色、荧光成像、信号切割和探针剥离的循环，许多不同的RNA物种可以在光学分辨率下被描绘出来。在应用这种方法时，我们证明了多重原位RNA分析可以在固定、冷冻和FFPE组织中成功实现。
Understanding the composition, regulation, and function of complex biological systems requires tools that quantify multiple transcripts at their native cellular locations. However, the current multiplexed RNA imaging technologies are limited by their relatively low sensitivity or specificity, which hinders their applications in studying highly autofluorescent tissues, such as formalin-fixed paraffin-embedded (FFPE) tissues. To address this issue, here we develop a multiplexed in situ RNA profiling approach with a high sensitivity and specificity. In this approach, transcripts are first hybridized by target-specific oligonucleotide probes in pairs. Only when these two independent probes hybridize to the target in tandem will the subsequent signal amplification by oligonucleotide hybridization occur. Afterwards, horseradish peroxidase (HRP) is applied to further amplify the signal and stain the target with cleavable fluorescent tyramide (CFT). After imaging, the fluorophores are chemically cleaved and the hybridized probes are stripped by DNase and formamide. Through cycles of RNA staining, fluorescence imaging, signal cleavage, and probe stripping, many different RNA species can be profiled at the optical resolution. In applying this approach, we demonstrated that multiplexed in situ RNA analysis can be successfully achieved in both fixed, frozen, and FFPE tissues.
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影响因子：
9.8
作者：
McQuin C;Goodman A;Chernyshev V;Kamentsky L;Cimini BA;Karhohs KW;Doan M;Ding L;Rafelski SM;Thirstrup D;Wiegraebe W;Singh S;Becker T;Caicedo JC;Carpenter AE
通讯作者：
Carpenter AE
影响因子：
5.5
作者：
Liao R;Mondal M;Nazaroff CD;Mastroeni D;Coleman PD;Labaer J;Guo J
通讯作者：
Guo J
影响因子：
48
作者：
Lubeck, Eric;Cai, Long
通讯作者：
Cai, Long
影响因子：
48
作者：
Raj, Arjun;van den Bogaard, Patrick;Rifkin, Scott A.;van Oudenaarden, Alexander;Tyagi, Sanjay
通讯作者：
Tyagi, Sanjay
影响因子：
18.3
作者：
Guo, Jia;Yu, Lin;Turro, Nicholas J.;Ju, Jingyue
通讯作者：
Ju, Jingyue