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

tion, the LightCycler FastStart DNA Master SYBR Green I Kit and LightCycler FastStart DNA Master Hybridization Probes Kit were used according to instructions: 0.5 M of each primer, 0.2 M HybProbe Probes and 4 mM MgCl2 were applied. The GFP-specific primers and HybProbe Probes were designed by the LightCycler Probe Design Software 1.0. Amplification was performed under the following conditions: a pre-incubation step for 10 minutes at 95C followed by 45 cycles of 10 seconds at 95C, 10 seconds at 60C, and 15 seconds at 72C (temperature ramp was 20C/seconds).

Results The human cell line PC3-GFP stably expresses GFP and shows a strong fluorescence signal (Figure 1A); 72 hours after transfection with chemically synthesized GFP-specific siRNA (GFP siRNA), the fluorescence signal was significantly reduced (Figure 1B).



The GFP knockdown was measured on the protein level. GFP expression was reduced by 53% (ng GFP/g total Protein) in PC3-GFP cells transfected with GFP siRNA compared with cells transfected with luciferase siRNA (Figure 2), which showed no GFP knockdown.



The LightCycler System is ideally suited for relative quantification of target mRNA levels in relation to reference gene mRNA levels. To determine the specificity of the used GFP siRNA on the mRNA level, a quantitative real-time PCR was performed using the LightCycler System.

In a first experiment, the different housekeeping gene expression levels in PC3-GFP cells were determined. The most suitable housekeeping gene was selected as a reference for relative quantification. GFP amplification and detection was performed with the LightCycler FastStart DNA Master SYBR Green I Kit, whereas the amplification and detection of
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