TransIT-TKO^® & TransIT-siQUEST^®

Fig. 1 | Knockdown Efficiencies Using TransIT-siQUEST^®, TransIT-TKO^®, Reagents and Lipofectamine^®, 2000. Firefly and sea pansy luciferase reporter vectors were co-transfected into various cell lines using the TransIT^®-LT1 Reagent (Mirus Bio). Subsequently, firefly luciferase expression was knocked down by transfection of 25 nM anti-firefly luciferase siRNA using either TransIT-siQUEST^® (red, Mirus Bio), TransIT-TKO^® (tan, Mirus Bio) or Lipofectamine^® 2000 (gray, Thermo Fisher Scientific) Reagents. Bars indicate the percent of normalized firefly luciferase expression as compared to each reagent alone control 24 hours post-transfection.

Fig. 2 | High Efficiency Endogenous Knockdown in iCell^® Cardiomyocytes. The TransIT-TKO^® Transfection Reagent was used to transfect iCell^® Cardiomyocytes (Cellular Dynamics International) plated at a density of 136,500 cells per well of a 12-well plate pre-coated with fibronectin. Seven days post-plating triplicate wells were transfected with TransIT-TKO^® (3-5 µl per well) and non-targeting control siRNA or GAPDH targeting siRNA (50nM per well). Seventy-two hours post-transfection, the amount of GAPDH mRNA was measured relative to 18s rRNA mRNA levels using qRT-PCR and then scaled to the expression level of the non-targeting control siRNA. Error bars represent the standard error of the mean (SEM) of three independent complexes.

Fig. 3 | High Efficiency Knockdown and Low Toxicity Using TransIT-TKO^® Reagent in HeLa Cells. Firefly and sea pansy luciferase reporter vectors were co-transfected into HeLa cells using TransIT^®-LT1 Reagent. Cells were incubated for at least 4 hours, split into 24-well plates, allowed to adhere and transfected with 25 nM of either a non-targeting siRNA or an anti-firefly luciferase siRNA using the indicated reagents with the volumes noted beneath each well. Luciferase expression, normalized to non-targeting siRNA control (bar graph) and lactate dehydrogenase (LDH) levels (line graph) were measured at 24 hours post-transfection. LDH levels are reported as % cytotoxicity compared to cells alone and were measured using a commercially available colorimetric assay; all values at or below zero are represented as zero on graph.

Fig. 4 | High Knockdown and Low Toxicity Using TransIT-siQUEST^® Reagent in CHO Cells Stably Expressing Firefly Luciferase. CHO-luc cells were grown in 24 wells plates and transfected with 25 nM of either a non-targeting siRNA or a anti-firefly luciferase siRNA using the indicated reagents with the volumes noted beneath each well. Luciferase expression, normalized to non-targeting siRNA control (bar graph) and lactate dehydrogenase (LDH) levels (line graph) were measured at 24 hours post-transfection. LDH levels are reported as percent cytotoxicity compared to cells alone and were measured using a commercially available colorimetric assay; all values at or below zero are represented as zero on graph.

Table 1 | Knockdown of Genes Using TransIT-TKO^® or TransIT-siQUEST^® Transfection Reagents. Cells were transfected with siRNAs targeting the indicated genes using the TransIT-TKO^® or TransIT-siQUEST^® Reagents (Mirus Bio), and the knockdown percentage was determined using quantitative RT-PCR or luciferase assays. *Firefly luciferase expression vectors were stably integrated into the parent cell lines and clonal lines constitutively expressing firefly luciferase were used.