Characterization of shRNA and shRNA interrupted by an intron in cultured cells. (A) The efficiency of the shRNA was analyzed by transiently transfecting Cos cells with vectors expressing firefly luciferase and Renilla luciferase. Firefly and Renilla luciferase activity was analyzed 48 h and 60 h after transfection using the Dual-luciferase Reporter Assay System in which firefly luciferase activity is normalized to Renilla luciferase activity. The pRNA-CMV3.1-Neo empty vector was used as a control, and the results are expressed as the mean ± S.D. of the percentage of control. (B) Silencing activity of the shRNA1-intron and shRNA2-intron in Cos cells 48 h and 60 h after transfection. (C) Cell viability was determined microscopically by trypan blue exclusion 48 h and 60 h after transfection with the pRNA-CMV3.1-Neo, shRNA1, shRNA1-intron, shRNA2 or shRNA2-intron vector. The total cell number and the viability were normalized by the values for cells transfected with the control vector and are expressed as the mean ± S.D. of the percent of control. (D) The efficiency of the shRNA and shRNA interrupted by the intron was also analyzed by transiently transfecting 293T cells and using the Dual-luciferase Reporter Assay System.