Highly Multiplexed Single-Cell In Situ RNA and DNA Analysis by Consecutive Hybridization.
Highly Multiplexed Single-Cell In Situ RNA and DNA Analysis by Consecutive Hybridization.
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DOI：
10.3390/molecules25214900
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发表时间：
2020-10-23
期刊：
影响因子：
--
通讯作者：
Guo J
中科院分区：
文献类型：
--
作者：
Xiao L;Liao R;Guo J
在自然空间环境中全面分析单个细胞中核酸的能力对于促进我们对生物学和医学的理解至关重要。在这里，我们报告了一种新的空间转录组学和基因组学分析的方法。在该方法中，在连续荧光原位杂交（C-FISH）的整个循环中，每个核酸分子在其天然细胞位置处被检测为荧光点。在每个C-FISH循环中，荧光寡核苷酸探针与前一循环中应用的探针杂交，并且还为下一循环探针引入结合位点。随着杂交、成像和光漂白的反复循环，各种核酸的身份由其独特的颜色序列确定。为了证明这种方法的可行性，我们表明，单细胞中的转录本或基因组位点可以用2个荧光团和16个C-FISH循环或3个荧光团和9个C-FISH循环明确定量。在没有任何误差校正的情况下，使用原始数据获得的误差率接近于零。这些结果表明，C-FISH可能使成千上万（216 = 65，536或39 = 19，683）的不同转录本或基因组位点能够在单个细胞中原位精确分析。
The ability to comprehensively profile nucleic acids in individual cells in their natural spatial contexts is essential to advance our understanding of biology and medicine. Here, we report a novel method for spatial transcriptomics and genomics analysis. In this method, every nucleic acid molecule is detected as a fluorescent spot at its natural cellular location throughout the cycles of consecutive fluorescence in situ hybridization (C-FISH). In each C-FISH cycle, fluorescent oligonucleotide probes hybridize to the probes applied in the previous cycle, and also introduce the binding sites for the next cycle probes. With reiterative cycles of hybridization, imaging and photobleaching, the identities of the varied nucleic acids are determined by their unique color sequences. To demonstrate the feasibility of this method, we show that transcripts or genomic loci in single cells can be unambiguously quantified with 2 fluorophores and 16 C-FISH cycles or with 3 fluorophores and 9 C-FISH cycles. Without any error correction, the error rates obtained using the raw data are close to zero. These results indicate that C-FISH potentially enables tens of thousands (216 = 65,536 or 39 = 19,683) of different transcripts or genomic loci to be precisely profiled in individual cells in situ.
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影响因子：
48
作者：
Chen F;Wassie AT;Cote AJ;Sinha A;Alon S;Asano S;Daugharthy ER;Chang JB;Marblestone A;Church GM;Raj A;Boyden ES
通讯作者：
Boyden ES
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：
56.9
作者：
Schrock, E;duManoir, S;Ried, T
通讯作者：
Ried, T
影响因子：
8
作者：
Olivo-Marin, JC
通讯作者：
Olivo-Marin, JC