Complete molecular analysis of target gene silencing: CSNK2A.

<p>HeLa cells were transfected with ten siRNA targeting CSNK2A, and two control siRNA, (GFP as a negative control and CSNK2B as a positive control) at a final concentration of 20 nM using Oligofectamine reagent. An additional control consisted in mock transfection of cells (without siRNA). Three days later, RNA and proteins were extracted for further analysis. <b>A.</b> Effect of siRNA treatment from a typical experiment. For each siRNA the relative quantity of the target mRNA to HPRT (black) or 36B4 (grey) was plotted using the comparative analysis module in MxPro software (Stratagene). <b>B.</b> Transfection efficiency control. For each experiment, transfection efficiencies were checked by quantifying gene silencing relative to a control siRNA of known efficiency. Results of experiments where this control did not silence expression by more than 70% were excluded from the dataset because transfection efficiency was considered to be poor. <b>C.</b> Box plot representation of siRNA efficiency for 10 sequences. For each siRNA, efficiency predicted by DSIR and measured efficiency are indicated. Measured efficiency was statistically determined from triplicate RT-qPCR quantification of target mRNA after siRNA treatment, based on three independent experiments. Expression levels were normalized to HPRT (black) and 36B4 (red) house-keeping genes. Log(Q) = 1 represents no reduction in target mRNA after treatment and log(Q) = 1/4 equates to approximately 75% efficiency. See section 2.6 for further details of the statistical analysis. Overall siRNA efficiency and significance values are provided in supplementary material. <b>D.</b> Western blot analysis of silencing efficiency, using anti-CK2alpha antibody. Protein loading was normalized for Hsp90 levels.