Silencing Efficacy Comparison between PepMute™ siRNA Transfection Reagent and Leading Products Figure 2. Excellent silencing of endogenously expressed KIF11 (also known as EG5) in HEK293 cells with 1.0 µl of PepMute™ reagent and 0.5 pmol EG5 siRNA per well of 24-well plate. KIF11 (also known as EG5) encodes a motor protein that belongs to the kinesin-like protein family involved in chromosome positioning and bipolar spindle formation during cell mitosis. A reduction in KIF11 levels causes mitotic arrest. PepMute™ reagent effectively delivers EG5 siRNA (final 1.0 nM) to HEK293 cells, leading to more than 80% of "round-up" phenotype of HEK293 cells 48h post transfection over negative control (final 1.0 nM with sham EG5 siRNA) while leading siRNA transfectin reagents, Lipofectamine™ RNAiMAX (RNAiMAX, 1.0 nM EG5 siRNA) / INTERFERin (1.0 nM EG5 siRNA) / Dharmafect (10.0 nM EG5 siRNA) and jetPRIME (20 nM EG5 siRNA) give average 37%, 23%, 53% and 48% ball-shaped phenotype respectively on HEK293 cells. The phenotype of "rounded-up" 293 cells were visualized (upper panel) and quantified (lower panel) 48h post transfection with a Nikon microscope.
Figure 3. Silencing efficiency comparison of PepMute™ Transfection Reagent (upper panel) with Dharmafect 4 (middle panel) and Lipofectamine™ RNAiMAX (RNAiMAX, lower panel) siRNA Transfection Reagents on A549 cells. siRNA targeting renilla luciferase at different final concentrations ranging from 0.5 to 20 nM was co-transfected with renilla luciferase gene (0.5 µg of pRL-CMV DNA per well) by the above three transfection reagents per manufacturers' protocols into A549 cells growing on a 24-well plate. Renilla luciferase activity was determined 36h after post co-transfection with renilla luciferase determination system (Promega). The luminescence was measured from 5.0 µl of lysate during 10s integration with a luminometer (Beckman Coulter LD 400). Luciferase activity was expressed as light units integrated over 10s (RLU) and normalized per mg of cell protein by using the BCA assay. The errors bars represent standard deviation derived from triplicate experiments. Luciferase-silencing efficiency was calculated relative to untreated cells. While PepMute™ and Dharmafect™ 4 reagents delivered significant gene silencing from 1.0 nM of renilla luciferase siRNA, Lipofectamine™ RNAiMAX gave good knockdown only after 20 nM while enhanced gene expression at low concentration of siRNA (0.5 and 1.0 nM respectively) was observed.
Figure 4. PepMute™ Transfection Reagent knocked down stable GFP expression in MCF7 cell (upper panel) and U2OS cell (lower panel) by reversely transfecting 5.0 and 1.0 nM GFP siRNA respectively. Green fluorescence protein (GFP) was stably expressed in MCF7 and U2OS cells. siRNA targeting GFP gene (right panel) and a sham siRNA (left panel) were introduced into MCF7 and U2OS cells with final 5.0 and 1.0 nM respectively by reverse transfection with PepMute™ Transfection Reagent. GFP gene silencing was monitored 48h post transfection by a Nikon fluorescence microscope. Quantitative analysis showed that GFP siRNA at 5.0 and 1.0 nM delivered by PepMute™ siRNA Transfection Reagent knocked down 90% and 95% stably expressed GFP in MCF7 and U2OS cells respectively.