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GENE SILENCING WITH PCR PRODUCTS

s Kits is a simple, three-step process: (I) one or two oligonucleotides encoding the siRNA sequence are designed and ordered; (II) the oligonucleotide(s) are used as primers in one or more PCRs with the included RNA Pol III promoter-containing DNA template; and (III) the resulting PCR product is column-purified (see Overview of Silencer Express Procedure for details of the procedure). The SECs are then transfected into cells using standard DNA transfection agents. Transfection conditions are similar, but not identical, to vectors and will require some optimization.

The 3' ends of the three different promoter elements provided in the Silencer Express Kits share a common twenty-nucleotide sequence (see RNA Pol III Promoter Structure for details). This domain serves as the priming site for appending siRNA templates during PCR. Because the sequence is common, a single siRNA template oligonucleotide can be used with any of the three Silencer Express Kits. The benefit is that different promoters can be tested for their capacity to express active siRNAs in the cells being used. Based on published reports, the activities of the different promoters are likely to vary from cell type to cell type (3). The localization of expressed RNA is also likely to vary with cell type and with the RNA pol III promoter used (3). To optimize siRNA expression, we find it beneficial to create SECs with at l
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