Single cell cytometry of protein function in RNAi treatedcells and in native populations
High Content Screening has been shown to improve results of RNAi and other perturbations, however significant variability continues to exist. Single cell cytometry can extract important information from subgroups within these samples.
Such approaches are important for immune cells analyzed by flow cytometry, but have not been broadly available for adherent cells that are critical to the study of solid-tumor cancers and other disease models.
Results: We have directly quantitated the effect of resolving RNAi treatments at single cell level in experimental systems for both exogenous and endogenous targets. Analyzing the effect of an siRNA that targets GFP at the single cell level permits a stronger measure of the absolute function of the siRNA by gating to eliminate background levels of GFP intensities.
Extending these methods to endogenous proteins, we have shown that well-level results of the knockdown of PTEN results in an increase in phospho-S6 levels, but at the single cell level, the correlation reveals the role of other inputs into the pathway. In a third example, reduction of STAT3 levels by siRNA causes an accumulation of cells in the G1 phase of the cell cycle, but does not induce apoptosis or necrosis when compared to control cells that express the same levels of STAT3.
In a final example, the effect of reduced p53 levels on increased adriamycin sensitivity for colon carcinoma cells was demonstrated at the whole-well level using siRNA knockdown and in control and untreated cells at the single cell level.
Conclusions: We find that single cell analysis methods are generally applicable to a wide range of experiments in adherent cells using technology that is becoming increasingly available to most laboratories.
It is well-suited to emerging models of signaling dysfunction, such as oncogene addition and oncogenic shock. Single cell cytometry can demonstrate effects on cell function for protein levels that differ by as little as 20%.
Biological differences that result from changes in protein level or pathway activate state can be modulated directly by RNAi treatment or extracted from the natural variability intrinsic to cells grown under normal culture conditions.
Author: Peter LaPan, Jing Zhang, Jing Pan, Andrew A Hill and Steven A Haney
Credits/Source: BMC Cell Biology 2008, 9:43
Published on: 2008-08-02
Copyright by the authors listed above - made available via BioMedCentral (Open Access). Please
make sure to read our disclaimer prior to contacting 7thSpace Interactive. To contact our editors, visit our online helpdesk. If you wish submit your own press release, click here.
Social Bookmarking
Digg this! | Post to del.icio.us | Post to Furl | Add to Netscape | Add to Yahoo! | Rojo
|
|
