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RESULTS
HeLa cells induced with PMA elicit a robust increase in the IL-8 expression. Based on the experimental results (data not shown), we determined that Cyclophilin B (PPIB) demonstrated more stable expression than GAPDH and subsequently was used as the normalization control. Following stimulation and siRNA transfection, the mRNA levels of each gene were measured from four replicate treatments of each experimental condition. The results are shown in Figure 2.
Coefficients of variation (CVs) measured from different treatments ranged from 5 to 15% over a 3-log range of relative luminescence (RLU) values. These CVs allow for the clear distinction of the effects of the siRNA seen at four hours (89% knockdown) from the effects seen at eight hours (94% knockdown). To normalize the results, the IL-8 and Cyclophilin B measurements made from the same replicate (i.e., the same batch of lysed cells) were converted to a ratio (IL-8 RLU to Cyclophilin B RLU). This ratio was calculated for each of the four replicates for each of the four experimental conditions. The results of this analysis are shown in Figure 3, where the error bars indicate one standard deviation. Following normalization, the range of CVs was determined to be 3 to 15%.
CONCLUSIONS
In the study described above, the QuantiGene Reagent System demonstrated a high level of precision and reproducibility in experiments that are commonly performed by researchers who utilize RNAi. CVs ranged from 5 to 15%, and from 3 to 15% when normalized to the expression of an endogenous control gene. The results of these experiments demonstrate that the QuantiGene Reagent System is a powerful companion t
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