t anti-mouse secondary antibody (a-MAP2) and Alexa Fluor 594-conjugated phalloidin, which binds to f-actin and serves here as an expression control.

Data kindly provided by A.M. Krichevsky and K.S. Kosik, Brigham and Womens Hospital, Harvard Medical School, Boston, MA (from Krichevsky, A.M., and Kosik, K.S. (2002) RNAi functions in cultured mammalian neurons. Proc. Natl. Acad. Sci. USA 99, 11926. Copyright 2002 National Academy of Sciences, U.S.A.)

Procedure All TransMessenger Reagent components are provided as ready-to-use solutions. To generate TransMessenger-RNA transfection complexes, simply mix your siRNA with Enhancer R and Buffer EC-R and incubate for 5 minutes at room temperature, then add TransMessenger Reagent and incubate for a further 10 minutes. The complexes are mixed with medium and added directly to the cells. Following a 3 hour incubation, the medium is changed and the cells are incubated until they are ready for analysis.

Optimal transfection results are achieved using high-purity siRNA. The QIAGEN siRNA Oligo Synthesis Service offers custom siRNA oligos, a range of library siRNAs that target specific genes, non-silencing control siRNAs, and a set of siRNAs targeting genes linked with cancer; the Cancer siRNA Oligo Set Version 1.0.
Since the amount of siRNA used is a critical factor for successful transfection, we recommend optimizing the amounts of siRNA and TransMessenger Transfection Reagent for every cell type-siRNA combination. To facilitate this, the reagent is provided with guidelines and starting points for optimization of RNAi experiments.

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