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

Transfection Reagent Handbook. siRNA (1 g per well) was condensed with Enhancer R and complexed with 4 l TransMessenger Reagent. The transfection complexes were diluted in 900 l of neurobasal medium and added directly to the cells. Medium containing transfection complexes was removed and replaced with neurobasal medium after 2 hours incubation. Cells were stained and analyzed after transfection. Double immunofluorescence labeling and image analysis was performed as described previously (1). siRNA sequences used to target endogenous MAP2 mRNA in neuronal cells Results and discussion The effect of siRNA-mediated RNAi on neuronal cells was tested using siRNA targeted against MAP2 endogenous mRNA. During the course of this study, it was noted that siRNA was more readily introduced into cells than plasmid DNA was. Cells were targeted with siRNA 58 days after plating. Double immunofluorescence labeling of MAP2 and the unrelated protein actin was performed 48 to 68 hours after transfection. The expression of MAP2 was specifically reduced in 7080% of cells, whereas the expression of an unrelated protein (f-actin) was unaffected (see Figure "Efficient Transfection of Primary Neuronal Cells Using QIAGEN siRNA and TransMessenger Reagent"). Transfection of either siRNA targeting MAP2 (siRNA1 or siRNA2) resulted in an identical level of inhibition of MAP2 expression. Efficient Transfection of Primary Neuronal Cells Using QIAGEN siRNA and TransMessenger Reagent Primary neuronal cells from
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