The Silencer GAPDH siRNA Control--and the TaqMan Gene Expression Assay for GAPDH--provide the ideal control siRNA and assay for optimization of transfection, respectively, (Figure 4).
Figure 3. Overview of the Process for siRNA Transfection Optimization.
Figure 4. Using qRT-PCR to Optimize Transfection of siRNA. Cos-7 cells were transfected with CDK2 siRNA or a negative control siRNA using the indicated volumes of transfection agent per well. 48 hr after transfection, the cells were harvested and analyzed by real-time RT-PCR using TaqMan Gene Expression Assays for CDK2 and 18S rRNA. Data from the 18S rRNA reactions were used to normalize input RNA, and the percent CDK2 expression was calculated as the amount of gene expression compared to the negative control siRNA.
Correlating Phenotype with the Extent of Knockdown
To better evaluate functional siRNA experimental results, the phenotype elicited should be correlated with the extent of knockdown induced by a particular siRNA. For a complete picture, both target mRNA levels and corresponding protein levels should be analyzed. Protein levels can be monitored by Western blot, immunofluorescence, ELISA or other means. (Protein can be isolated using Ambion's PARIS Kit.) For quantitating mRNA levels, however, qRT-PCR is again the preferred choice.