Efficient Selection of Silenced Primary Cells by Flow Cytometry

Background: RNA interference has emerged as a new and potent tool to knockdown the expression of target genes and to investigate their functions. For short time experiments with mammalian cell lines, RNA interference is typically induced by transfecting small interfering RNAs (siRNAs). Primary cells constitute important experimental systems in many studies because of their similarity to their in vivo counterparts; however, transfection of these cells has been found to be difficult. As a consequence, RNA interference of primary cells may result in mixed phenotypes because of the simultaneous presence in the same preparation of transfected and nontransfected cells. This may be particularly inconvenient when certain experiments (for example, biochemical analysis) should be performed. Methods: We use fluorescently labeled siRNAs to induce RNA interference in fibroblasts, and flow-cytometry associated cell sorting to separate subpopulations of transfected cells according to fluorescence intensity. Results: Flow cytometry allows one to discriminate between strongly- and weakly- or nonsilenced fibroblasts, since the fluorescence intensity of transfected cells is related to the number of internalized siRNA copies and to the mRNA knockdown efficiency. Conclusions: The use of fluorescently labeled siRNAs may allow one to isolate by flow-cytometry associated cell sorting the most efficiently silenced primary cells for subsequent analysis. © 2007 International Society for Analytical Cytology