Quantitative and temporal analysis of gene silencing in tumor cells induced by small interfering RNA or short hairpin RNA expressed from plasmid vectors.

Vector-based RNA interference (RNAi) has attracted great interest, because of its more prolonged gene silencing effect compared with small interfering RNA (siRNA). However, the intensity and duration of vector-based RNAi effect has received little attention. In this study, the gene silencing kinetics of short hairpin RNA (shRNA)-expressing plasmid DNA (pDNA) driven by U6, H1 or tRNA promoter (pU6-shLuc, pH1-shLuc, and ptRNA-shLuc) was studied in melanoma cells expressing firefly luciferase. A bootstrap method-based moment analysis was performed to statistically and quantitatively evaluate the profile of gene silencing. The analysis showed that pU6-shLuc induced a significantly greater and longer gene silencing than that produced by other promoter-driven shRNA expression vectors. In addition, it was found that pU6-shLuc was at least 100-fold more potent in gene silencing than siRNA targeting the same gene on a numerical basis. These statistical considerations demonstrated that U6 promoter-driven shRNA expressing pDNA is the most effective in inducing gene silencing effect as far as the intensity and duration of RNAi effect is concerned. (c) 2008 Wiley-Liss, Inc. and the American Pharmacists Association