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Efficient RNAi-mediated gene silencing in neuronal cells using QIAGEN ,,, siRNA and TransMessenger Transfection Reagent*

rats were transfected with a control nonspecific siRNA or an siRNA directed against MAP2 using TransMessenger Transfection Reagent. Cells were stained 4868 hours after transfection using mouse monoclonal anti-MAP2 antibodies and Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (MAP2) and Alexa Fluor 594-conjugated phalloidin, which binds to f-actin and serves here as an expression control. Suppression of MAP2 Leads to Defective Neuronal Development Primary neuronal cells from rats were transfected with an siRNA directed against MAP2 using TransMessenger Transfection Reagent. Primary cultures were targeted with siRNA 3 hours after plating and analyzed over 2840 hours. Cells were stained using mouse monoclonal anti-MAP2 antibodies and Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (MAP2) and Alexa Fluor 594-conjugated phalloidin. The cell with silenced MAP2 expression (arrowed) exhibits severely defective filopodial elaboration.

MAP2 has been shown to be required to overcome early transitions of neuritic development and to begin elongation of neuritic processes (2, 3). Phenotypic effects of MAP2 suppression can be observed early on after plating so primary neuronal cultures were targeted with siRNA 3 hours after plating and the effect was analyzed over 28 to 40 hours. Within the shorter time frame, fewer neurons suppressed MAP2 expression, however the degree of MAP2 suppression correlated with a recognized defect in filopodia elaboration (see Figure "Suppression of MAP2 Leads to Defective Neuronal Development").

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