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Quantification of siRNA Silencing Efficiency,,,Using the LightCycler System

the housekeeping genes were carried out using the LightCycler h-Housekeeping Gene Selection Set and the LightCycler FastStart DNA Master Hybridization Probes Kit. The data from the housekeeping genes were used to normalize the GFP results (Figure 3). Of the five tested housekeeping genes (β2M, G6PDH, ALAS, HPRT and PBGD) the expression level of β2Microglobulin (β2M) was closest to the expression level of GFP (Figure 3a). Therefore it was selected as reference for relative quantification in the second experiment. The down-regulation of GFP mRNA was in the same range, independent of the housekeeping gene used as a reference for relative quantification. GFP mRNA was down-regulated by 81%89% by GFP siRNA in comparison to luciferase siRNA, which showed no down-regulation (Figures 3a, b).



In a second experiment, the GFP knockdown was detected using HybProbe Probes. The resulting quantification data were normalized with the β2M quantification data. The amplification of the GFP cDNA showed a strong reduction of GFP mRNA in PC3-GFP cells transfected with GFP siRNA compared to PC3-GFP cells transfected with luciferase siRNA (Figure 4b). GFP mRNA was down-regulated by 85% with GFP siRNA compared with luciferase siRNA. In contrast, the expression level of the housekeeping gene in PC3-GFP cells transfected with luciferase siRNA, or with GFP siRNA was nearly identical (Figure 4a).



Conclusion

To confirm the specificity of a down-regulation caused by siRNA, it is necessary to show that the down-regulation on the protein level can also be detected on the mRNA level. Especially for very stable proteins like GFP it is often difficult to see a high knockdown on protein level. Using the LightCycler System, we could show
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