Messenger RNA quantification after fluorescence-activated cell sorting using in situ hybridization

Recent studies using stem cells or cancer stem cells have revealed the importance of detecting minor populations of cells in blood or tissue and analyzing their biological characteristics. The only possible method for carrying out such procedures is fluorescence-activated cell sorting (FACS). However, FACS has the following two limitations. First, cells without an appropriate cell surface marker cannot be sorted. Second, some laborious procedures such as rapid sorting or treatment under sterilized conditions may require in order to analyze their biological characteristics. If a specific mRNA in a particular cell type can be stained with a florescent dye and then the cells can be sorted without causing RNA degradation, a more simple and universal method for sorting and analyzing cells with a specific gene expression pattern could be established since the biological characteristics of the sorted cells could then be determined by analyzing their gene expression profile. In this study, we established a basic protocol for messenger RNA quantification after FACS (FACS-mQ) using a cRNA probe. This method could be used for the detection and analysis of stem cells or cancer stem cells in various tissues. © 2010 International Society for Advancement of Cytometry