According to the data obtained, the inhibition of 506 siRNA was 39.2%, while those of 859 siRNA and 891 siRNA were 89.4% and 54.1%, respectively (Fig. 6). The best interference effect was observed on 859 siRNA, up to 89.4% of inhibition rate. Ishii et al. [10] reported that MeWo fibroblast cell (BCRC 60540), a kinds of human melanoma cells, can express spontaneously the endogenous

selleckchem MMP1 protein, and the expression quantity could be enhanced by exposure to nitric oxide (NO) or S-nitroso-N-acetyl-dl-penicillamine (SNAP) in a dose-dependent manner. Therefore, the MeWo cells were employed as target cells in quantitative PCR and western blot analysis to investigate whether the evaluation of effective designed siRNAs by the GFP reporter systems was able to interfere with the expression of MMP1 mRNA or protein. According to the results of preliminary experiments, Fig. 7 and Fig. 8, , the expression quantity and the siRNA interfering efficiency of endogenous mRNA or protein of MMP1 in MeWo cells without induction by NO or SNAP was visible in the blanks (without siRNA treatment). Accordingly, the MeWo cells used in this study were not treated with NO or SNAP to

avoid influence by other factors. CP 868596 To confirm the interference efficacy of siRNAs against endogenous MMP1 gene expression in MeWo cells, the quantitative PCR (real-time PCR) was prepared. The MeWo cells were transfected with various concentrations (10, 30, 50, 70 or 90 nM) of 506 siRNA, 859 siRNA and 891 siRNA, separately. The total RNA was extracted and cDNA were synthesized as described in the methodology. The remained MMP1 mRNA was analyzed by real-time quantitative polymerase chain reaction (RT-PCR) and agarose gel electrophoresis analysis. According to the results of preliminary experiments, when the concentration of any one of the three

designed siRNAs was higher than 100 pmol, the interference efficacy was not consistent and had high standard deviation between repetitive experiments (data not shown). This might be attributed to short half-life and instability of siRNAs. The phenomenon was similar to [11], they had suggested that when the concentration of siRNA was higher than 100 nM, it could cause off-target new effect, resulting in the error judgments of the experimental results, so in this study, all the concentration of siRNA used in different tests was ranged from 10 to 90 nM. As shown in Fig. 7, the endogenous MMP1 mRNA could be interfered with 506 siRNA and 859 siRNA, but the interference efficacy of various concentrations of siRNAs on endogenous MMP1 gene expression were not dose-dependent. The inhibition rates of 506 siRNA and 859 siRNA against endogenous MMP1 gene expression were 55% and 85%, respectively (Fig. 7).