Figure 2.

Combination of RNAi and U1i increases luciferase knockdown in vitro. (a) Co-transfection of 50 ng control shApoB construct or 50 ng shLuc1 construct with 200 ng (or 100+100 ng) U1i plasmid and firefly luciferase and renilla luciferase plasmid. Transfections and analysis were carried out as described in Figure 1a. Cells transfected with a combination of 50 ng shApoB and 200 ng U1C were set at 100%. Co-transfection of shApoB with L4 and L5 or L4/L5 significantly reduced luciferase activity by ∼65% compared with the 100% control (P<0.05). Transfection of shLuc with U1C resulted in a significant 86% knockdown (P<0.001). (b) Co-transfection of 50 ng shLuc1 construct with 200 ng (or 100+100 ng) U1i plasmid and firefly luciferase and renilla luciferase. Note the different scales on the y axes in (a, b). Co-transfection of shLuc1 with L4 and L5 maximally reduced luciferase expression by 95%. Control U1i constructs were U1C, and H1 and H4 that target the human huntingtin sequence. Luciferase activity after co-transfection of shLuc1 with L4 and L5 or L4/L5 was significantly different from co-transfection of shLuc1 with U1C (P<0.05), and from co-transfection of shLuc1 with H1 and H4 (P<0.001) or H1/H4 (P<0.05). Data are presented as mean of three independent experiments +s.d., analyzed using factor correction.³⁶