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

Anna M. Krichevsky and Kenneth S. Kosik
Center for Neurologic Diseases, Brigham and Womens Hospital, Harvard Medical School, Boston, MA, USA Genetic manipulations using neuronal cells are notoriously difficult. Therefore, efficient delivery of short interfering RNA (siRNA) to primary neuronal cultures is of critical importance for RNA interference (RNAi)-mediated gene suppression. Here we describe successful targeted suppression of gene expression using TransMessenger Transfection Reagent and synthetic siRNA directed against microtubule-associated protein 2 (MAP2). RNAi has become a powerful tool used to knock down the expression of genes in cultured mammalian cells. However, neuronal cells, which are difficult to manipulate, have seemed to be more resistant to RNAi than other cell types. The reasons for this are unclear. Differences in siRNA uptake across the neuronal cell membrane or the RNAi pathway itself are possible causes. In this study, synthetic siRNA was used to successfully suppress expression of MAP2.

Materials and methods Two QIAGEN siRNAs targeted against MAP2 mRNA were designed with a 5' phosphate, 3' hydroxyl, and two-base overhangs on each strand (see Table "siRNA sequences used to target endogenous MAP2 mRNA in neuronal cells"). Transfections of siRNA for gene targeting were performed using TransMessenger Transfection Reagent as described in the TransMessenger
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