Detection of Low Abundance RNA Molecules in Individual Cells by Flow Cytometry
Detection of Low Abundance RNA Molecules in Individual Cells by Flow Cytometry
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DOI：
10.1371/journal.pone.0057002
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发表时间：
2013-02-18
期刊：
影响因子：
3.7
通讯作者：
Park, Emily
中科院分区：
文献类型：
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作者：
Hanley, Mary Beth;Lomas, Woodrow;Park, Emily
多种RNA分析技术可用于检测来自大量细胞群体的RNA转录物。然而，从单个细胞中检测RNA的技术受到限制。在这里，我们采用了一种新的原位信号放大方法（RNAScope（R）检测平台），通过流式细胞术分析单个细胞中的细胞内RNA。使用新的目标特异性探针，旨在抑制背景信号，我们证明了在HIV感染的细胞样本中的HIV gag RNA的特异性检测，除了在K562细胞系中的bcr和abl mRNA。这种方法能够区分表达低丰度RNA转录本的细胞，并与定量成像分析相关性良好。此外，多个不同的RNA靶标以高特异性同时检测而不受干扰。总的来说，该方法的灵敏度和特异性将有助于分析来自单个细胞的功能重要的RNA种类，即使在非常低的拷贝数下。
A variety of RNA analysis technologies are available for the detection of RNA transcripts from bulk cell populations. However, the techniques for RNA detection from individual cells have been limited. Here we adapt a novel in situ signal amplification method (the RNAScope (R) detection platform) for the analysis of intracellular RNAs in individual cells by flow cytometry. Using novel target-specific probes that were designed to suppress background signals, we demonstrate the specific detection of HIV gag RNAs in HIV-infected cellular samples, in addition to bcr and abl mRNAs in the K562 cell line. This method was capable of distinguishing cells expressing low abundance RNA transcripts and correlated well with quantitative imaging analysis. Furthermore, multiple distinct RNA targets were simultaneously detected with a high specificity without interference. Overall, the sensitivity and specificity of this method will be useful for the analysis of functionally important RNA species from individual cells, even at very low copy numbers.