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

✍ Scribed by Gloria Juan; Eva Hernando; Carlos Cordon-Cardo

• Publisher: John Wiley and Sons
• Year: 2002
• Tongue: English
• Weight: 463 KB
• Volume: 49
• Category: Article
• ISSN: 0196-4763
• DOI: 10.1002/cyto.10173

✦ Synopsis

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 (G~1~ and G~2~/M) to obtain good quality RNA for gene expression analyses.

Methods

The experimental design included sorting G~1~ and G~2~/M cells with the vital fluorochrome Hoechst 33342, followed by RNA isolation from the sorted cells.

Results

Sorted living G~1~ and G~2~/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. © 2002 Wiley‐Liss, Inc.