Monitoring differential synthesis of RNA in individual cells by capillary electrophoresis.
A capillary electrophoresis (CE)-based technique is reported here to monitor differential RNA synthesis in individual Chinese hamster ovary cells at distinct stages of the cell proliferation cycle. Cell synchronization was achieved by the shake-off method, in which mitotic (M) cells were dislodged, and cells at G(1), S, and G(2) phases were harvested 2.5, 10, and 13 h, respectively, after synchronizing the mitotic cells. Thirty-two cells (eight from each phase) were analyzed by injecting each cell into the capillary, lysing it with dilute surfactant, separating the RNA by capillary electrophoresis, and detecting the peaks with laser-induced fluorescence. The results from single cells show that the total amount of RNA increased at each successive stage (from G(1) to M), while the relative synthetic rates of different RNA fractions varied with progression through the cycle. There was a threefold increase in the synthetic rate of total RNA from S to G(2), compared with G(1) to S. In addition, differential accumulation of specific RNA fractions was observed, with the low-molecular-mass fraction exhibiting a much higher synthetic rate from G(2) to M, relative to the rates of the larger ribosomal RNA (rRNA) fractions. Comparison of the large rRNA fractions with one another reveals that at S phase more 28S rRNA was accumulated than 18S rRNA, and at G(1) and M phases, the synthetic rate of 28S rRNA was slowed compared with that of 18S. Minimal sample preparation, combined with the separation power of CE and single-cell detection sensitivity of laser-induced fluorescence, results in a simple method for assessing differential accumulation of RNA from distinct individual cells.
Han
Futian
F
Department of Chemistry, University of California, Riverside, California 92521, USA.
Lillard
Sheri J
SJ
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D019075:Electrophoresis, Capillary / Q000379:methods*; D012313:RNA / Q000096:biosynthesis*
D012313:RNA