[FACS-mQ as a powerful clinical test in the cancer stem cell era]

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.