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More siRNA Vectors for RNA Interference

the rapid and inexpensive preparation of vectors that can be used to select the RNA Pol III promoter that is best suited for your experimental protocol.

Using the pSilencer vectors, Ambion scientists have reduced the expression of GAPDH, cyclophilin, c-myc, p53, and cdk-2 (RNA and protein levels). An example experiment is shown in Figure 2. As expected, the two pSilencer plasmids are not equally effective at reducing target gene expression (Figure 2). This is likely due to differential siRNA expression levels or siRNA localization in the cells that we tested.

Figure 2. pSilencer-Induced Reductions in Target Gene Expression. pSilencer 2.0-U6 and 3.0-H1 vectors encoding hairpin siRNAs specific to GAPDH, cyclophilin, or a non-genomic sequence were transfected into HeLa cells. Forty-eight hours post-transfection, target RNA and protein levels were assessed. (A) 1 ug of total RNA isolated from various cell samples was assessed by Northern analysis using the NorthernMax procedure (Ambion). RNA probes specific to GAPDH, cyclophilin, and 28S rRNA were used to probe the Northerns. The specific activity of the 28S rRNA probe was approximately 100,000-fold lower than the mRNA-specific probes. (B) GAPDH protein levels in cells transfected with pSilencer 2.0-U6-GAPDH were analyzed by immunofluorescence using a GAPDH-specific antibody (Ambion). Green: anti-GAPDH antibody detected with fluorescein la
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