Gene silencing by 2nd generation shRNA is effective and sequence-specific. (A) The effect of wt and 2nd generation shRNA inhibitors on a luciferase reporter gene with the HIV-1 target sequence (wt, G8A or G15A). 293T cells were co-transfected with 25 ng firefly luciferase reporter plasmid (wt, G8A or G15A), 0.5 ng of renilla luciferase plasmid, and 0, 1 and 5 ng shRNA constructs. Relative luciferase activity were determined as the ration of the firefly and renilla luciferase expression. Values are shown as percentage of the transfection without shRNA. Averages and standard deviations represent at least three independent transfections that were performed in quadruple.