Streamline Your siRNA Transfections siPORT™ NeoFX™ Transfection Agent Promotes High Reproducibility and Efficiency
Streamlined Transfection Protocol
Figure 1. Traditional Plated Transfection vs. Neofection with siPORT™ NeoFX™
Optimized for Lower siRNA Concentrations
Figure 2. Efficient Transfection with Low siRNA Concentration. HeLa cells were trypsinized and resuspended in growth media at a concentration of 4x104 cells/ml. Transfection complexes were prepared using chemically synthesized GAPDH siRNA (0.03 nM to 10 nM) or negative control siRNA (data not shown) and 0.5 µl siPORT™ NeoFX™ Transfection Agent. 48 hours after transfection, cells were harvested and analyzed by real-time RT-PCR for both GAPDH mRNA and 18S rRNA levels. Percent mRNA remaining was calculated as the amount of GAPDH mRNA in cells transfected with GAPDH siRNA compared to that of cells transfected with the negative control siRNA. Data were normalized to the 18S rRNA signal. These results demonstrate that siPORT NeoFX performs well even with reduced levels of siRNA, which may offer significant benefits for eliminating off-target effects in gene-silencing applications.
High siRNA Transfection Efficiency with Low Cytotoxicity
Figure 3. High Efficiency and Low Cytotoxicity of siPORT™ NeoFX™ Transfection Agent. (A) HeLa cells were transfected with 30 nM Cy™3-labeled siRNA using siPORT NeoFX Transfection Agent. Cells were subsequently fixed, stained with DAPI for visualization of nuclei, and analyzed by fluorescence microscopy. Blue: DAPI stained nuclei; Red: Cy3 fluorescent siRNA. (B) CDK2 siRNA or Silencer™ Negative Control siRNA #1 was complexed with six different transfection agents according to manufacturer's instructions and added to empty wells of a 96 well dish (final siRNA concentration of 30 nM). HepG2 cells were added to the wells and cultured for 48 hours in complete media containing serum. Cell viability was analyzed by microflow cytometry using PCA 96 instrument and Viacount™ assay (Guava Technologies). RNA was isolated, and target gene expression was quantified by real time RT-PCR. Relative reduction in mRNA levels is expressed as a percentage of expression in cells transfected with a negative control siRNA. Duplicate samples were normalized by measuring 18S rRNA levels by real time RT-PCR. The range of normalized gene expression is shown with error bars.
Lot-to-lot, Day-to-day, Plate-to-plate, and Well-to-well Consistency in Performance
Figure 4. Consistent Day-to-day, Plate-to-plate, and Well-to-well Performance. 8,000 HeLa cells were transfected in 96 well plates using siPORT™ NeoFX™ (0.3 µl/100 µl reaction volume) and either GAPDH siRNA (10 nM) or a scrambled negative control siRNA. Remaining GAPDH expression was quantified as described in Figure 2. Each bar represents the mean of eight replicate wells. siPORT NeoFX demonstrates consistent transfection performance across multiple wells, among multiple plates, and on multiple days.