Separation of live cells in different phases of the cell cycle for gene expression analysis

Open Access
Publisher Website
Google Scholar
Abstract
Background Homogeneity of cell populations is a basic requirement for gene expression analyses of the cell cycle, such as those based on microarrays. The most common approach to obtain specific populations is the use of synchronization methods that increase the number of cells representing a certain cell cycle stage. On the one hand, conventional synchronization usually causes undesirable effects. On the other hand, cell separation methods may imply loss of RNA quality, another limiting factor for expression profiling. We describe a new strategy to specifically separate live cells in different phases of the cell cycle (G1 and G2/M) to obtain good quality RNA for gene expression analyses. Methods The experimental design included sorting G1 and G2/M cells with the vital fluorochrome Hoechst 33342, followed by RNA isolation from the sorted cells. Results Sorted living G1 and G2/M cells, analyzed by immunocytochemistry and laser scanning cytometry, showed strong enrichment. The quality and specificity of the isolated RNA were demonstrated by northern blot. Conclusions This new approach has many potential applications, such as expression profiling of specific cell populations after eliminating the irrelevant data produced by cells in other stages of the cycle. Cytometry 49:170–175, 2002.