Separation of live cells in different phases of the cell cycle for gene expression analysis.
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.
Juan
Gloria
G
Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
Hernando
Eva
E
Cordon-Cardo
Carlos
C
D001562:Benzimidazoles / Q000378:metabolism; D002295:Carcinoma, Transitional Cell / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D002453:Cell Cycle / Q000235:genetics*; D002469:Cell Separation; D019926:Cyclin B / Q000235:genetics / Q000378:metabolism; D056744:Cyclin B1; D004273:DNA, Neoplasm / Q000032:analysis; D005434:Flow Cytometry; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D007150:Immunohistochemistry; D012333:RNA, Messenger / Q000378:metabolism; D012334:RNA, Neoplasm / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
D001562:Benzimidazoles; C534699:CCNB1 protein, human; D019926:Cyclin B; D056744:Cyclin B1; D004273:DNA, Neoplasm; D012333:RNA, Messenger; D012334:RNA, Neoplasm; C017807:bisbenzimide ethoxide trihydrochloride