Takano Toru, Hidaka Yoh, Iwatani Yoshinori
Department of Laboratory Medicine, Osaka University Graduate School of Medicine, Suita 565-0871, Japan.
Rinsho Byori. 2012 Aug;60(8):748-52.
Stem cells are pluripotent and self renewing, and possess an ability to differentiate into the various cell types of a particular tissue. In cancer tissues, existence of cells showing biological similarities with stem cells, named cancer stem cells (CSC), are known to regulate the growth of the tissue and determine its prognosis. Stem cells and CSCs usually exist as minor populations of cells in a tissue. Detection and analysis of these cells are usually laborious even using fluorescence activated cell sorting (FACS) with the conventional protocols. Considering these drawbacks, we developed a novel analytical method named mRNA quantification after FACS (FACS-mQ). In FACS-mQ, cells are labeled with a fluorescent dye in a manner that minimizes RNA degradation, then cells sorted by FACS are examined by analyzing their gene expression profile. We established protocols to obtain single cells from clinical samples for flow cytometry analysis. Further, we performed FACS-mQ analysis using fluorescence-labeled antibodies, cRNA probes and locked nucleic acid (LNA) probes. Evident decrease of intracellular RNAs did not observed in FACS-mQ using immunocytochemistry. Approximately 60% of intracellular RNA was preserved after in situ hybridization using cRNA probes. These RNAs from a small number of sorted cells were suitable for quantitative analysis to establish gene expression profiles. FACS-mQ does not require laborious and time-consuming procedures; thus, it is expected to facilitate research on stem cells or cancer stem cells.
干细胞具有多能性且能自我更新，具备分化为特定组织中各种细胞类型的能力。在癌组织中，已知存在与干细胞具有生物学相似性的细胞，即癌症干细胞（CSC），它们可调节组织生长并决定其预后。干细胞和癌症干细胞通常在组织中以少数细胞群体的形式存在。即便使用传统方案通过荧光激活细胞分选（FACS）来检测和分析这些细胞，通常也很费力。考虑到这些缺点，我们开发了一种名为FACS后mRNA定量分析（FACS-mQ）的新型分析方法。在FACS-mQ中，细胞用荧光染料标记，标记方式可使RNA降解最小化，然后通过分析FACS分选的细胞的基因表达谱来对其进行检测。我们建立了从临床样本中获取单细胞用于流式细胞术分析的方案。此外，我们使用荧光标记抗体、cRNA探针和锁核酸（LNA）探针进行了FACS-mQ分析。在使用免疫细胞化学的FACS-mQ中未观察到细胞内RNA明显减少。使用cRNA探针进行原位杂交后，约60%的细胞内RNA得以保留。这些来自少量分选细胞的RNA适用于定量分析以建立基因表达谱。FACS-mQ不需要费力且耗时的程序；因此，有望促进对干细胞或癌症干细胞的研究。
