mRNA Quantification After Fluorescence Activated Cell Sorting Using Locked Nucleic Acid Probes
mRNA Quantification After Fluorescence Activated Cell Sorting Using Locked Nucleic Acid Probes
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DOI：
10.1007/s12033-011-9375-9
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发表时间：
2011-09-01
影响因子：
2.6
通讯作者：
Takano, Toru
中科院分区：
文献类型：
--
作者：
Maruo, Rie;Yamada, Hiroya;Takano, Toru
最近，我们已经建立了一种管中原位杂交方法，称为荧光激活细胞分选后mRNA定量（FACS-mQ），其中特定细胞类型中的特定RNA用荧光素染料染色，允许通过FACS选择染色的细胞而不遭受过度的RNA降解。使用这种方法，可以通过分析其基因表达谱来确定分选细胞的生物学特性。在这项研究中，我们使用锁核酸（LNA）寡核苷酸，这是已知的，以提高RNA检测的灵敏度和特异性，作为杂交探针在FACS-mQ。当我们使用靶向人类28 S序列的LNA探针时，我们能够有效地将人类细胞与大鼠细胞分离。使用LNA探针，杂交步骤缩短至1小时。在杂交步骤后，84.6%的RNA被保留;因此，我们能够在FACS后成功地测量每种类型细胞中的基因表达水平。提供LNA探针与靶序列有效杂交，具有LNA探针的FACS-mQ是用于分离特定细胞并通过分析其基因表达谱来确定其生物学特性的有力工具。
Recently, we have established an in-tube in situ hybridization method named mRNA quantification after fluorescence activated cell sorting (FACS-mQ), in which a specific RNA in a particular cell type is stained with a florescent dye, allowing the stained cells to be selected by FACS without suffering excessive RNA degradation. Using this method, the biological characteristics of the sorted cells can be determined by analyzing their gene expression profile. In this study, we used locked nucleic acid (LNA) oligonucleotides, which are known to enhance both the sensitivity and specificity of RNA detection, as hybridization probes in FACS-mQ. When we used a LNA probe targeting the human 28S sequence, we were able to efficiently separate human cells from rat cells. Using LNA probes, the hybridization step was shortened to 1 h. After the hybridization step, 84.6% RNA was preserved; thus, we were able to successfully measure gene expression levels in each type of cell after FACS. Providing the LNA probe efficiently hybridizes with the target sequence, FACS-mQ with an LNA probe is a powerful tool for separating particular cells and determining their biological characteristics by analyzing their gene expression profile.