FISH-Flow, a protocol for the concurrent detection of mRNA and protein in single cells using fluorescence in situ hybridization and flow cytometry.
FISH-Flow, a protocol for the concurrent detection of mRNA and protein in single cells using fluorescence in situ hybridization and flow cytometry.
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DOI：
10.1038/nprot.2017.039
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发表时间：
2017-06
期刊：
影响因子：
14.8
通讯作者：
Gennaro ML
中科院分区：
文献类型：
--
作者：
Arrigucci R;Bushkin Y;Radford F;Lakehal K;Vir P;Pine R;Martin D;Sugarman J;Zhao Y;Yap GS;Lardizabal AA;Tyagi S;Gennaro ML
我们描述了一种基于流式细胞术的方法，用于使用荧光原位杂交(FISH)探针测量非贴壁哺乳动物细胞内的mRNA。这种方法，我们称之为FISH-FLOW，允许高通量多参数测量基因表达，这是一项用早期基于显微镜的方法不可行的任务。FISH-FLOW方案包括细胞固定、渗透和与一组荧光标记的寡核苷酸探针杂交。在这个方案中，表面和细胞内的蛋白质标记也可以用荧光标记的抗体进行染色，以同时进行蛋白质和mRNA的测定。此外，该方案的半自动、单管版本可以使用商业上可获得的细胞清洗设备来执行，该设备可以减少细胞损失、操作员时间和操作员间的可变性。执行该方案需要大约30小时。描述了通过体外刺激具有特定抗原的预置T细胞来诱导细胞因子mRNA的FISH-FLOW测量实例。
We describe a flow-cytometry-based protocol for intracellular mRNA measurements in nonadherent mammalian cells using fluorescence in situ hybridization (FISH) probes. The method, which we call FISH-Flow, allows for high-throughput multiparametric measurements of gene expression, a task that was not feasible with earlier, microscopy-based approaches. The FISH-Flow protocol involves cell fixation, permeabilization and hybridization with a set of fluorescently labeled oligonucleotide probes. In this protocol, surface and intracellular protein markers can also be stained with fluorescently labeled antibodies for simultaneous protein and mRNA measurement. Moreover, a semiautomated, single-tube version of the protocol can be performed with a commercially available cell-wash device that reduces cell loss, operator time and interoperator variability. It takes ~30 h to perform this protocol. An example of FISH-Flow measurements of cytokine mRNA induction by ex vivo stimulation of primed T cells with specific antigens is described.
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影响因子：
3.7
作者：
Hanley, Mary Beth;Lomas, Woodrow;Park, Emily
通讯作者：
Park, Emily
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
DOI：
10.1007/978-1-62703-526-2_3
发表时间：
2013-01-01
期刊：
IMAGING GENE EXPRESSION: METHODS AND PROTOCOLS
影响因子：
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作者：
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通讯作者：
Zenklusen, Daniel
影响因子：
9.9
作者：
Shah, Khyati;Tyagi, Sanjay
通讯作者：
Tyagi, Sanjay
影响因子：
56.9
作者：
Femino, A;Fay, FS;Singer, RH
通讯作者：
Singer, RH