• Fast—save a day by transfecting cells as they are plated
  • Efficient—high performance delivery allows use of low siRNA concentrations
  • Reproducible—consistent results, lot-to-lot, plate-to-plate, and well-to-well
  • Versatile—works with a broad range of cell lines
  • Powerful—performs with low cytotoxicity in the presence or absence of serum

Conducting and optimizing gene silencing experiments can be time consuming, due in part to the need to pre-plate cells the day before transfection. However, siPORT™ NeoFX™ Transfection Agent was developed to streamline siRNA transfection experiments. This lipid-based formulation efficiently transfects adherent cells as they are subcultured—without increased cytotoxicity. Successful gene silencing experiments with siPORT NeoFX Transfection Agent can be completed in less than 24 hours. This protocol can be adapted to a wide range of cell lines (Figure 1) and experimental designs, including high throughput applications.

Save a Day by Transfecting Cells during Plating

Although compatible with standard transfection methods, siPORT NeoFX Transfection Agent can be used to transfect cells during subculturing. Just mix this transfection agent with siRNA, incubate to form transfection complexes, add to culture wells, and overlay with cells. This protocol saves a day compared to traditional plated transfection procedures. In addition, there is no need to remove or replace media following transfection, because the transfection complexes are active and stable even in the presence of serum.

High Performance Delivery Allows Use of Low siRNA Concentrations

siRNA concentrations ≥100 nM can lead to nonspecific changes in gene expression (off-target effects) in mammalian cultured cells. Reducing the amount of siRNAs used for transfections to 1–20 nM minimizes these nonspecific effects, while still providing effective silencing of the target gene. siPORT NeoFX Transfection Agent has been optimized to deliver even picomolar amounts of siRNA (Figure 2).

Obtain Consistent Results

siPORT NeoFX Transfection Agent is remarkably stable at various temperatures, which results in reproducible siRNA delivery from day to day and experiment to experiment. All of these favorable properties make this transfection agent an effective reagent for RNAi screening experiments.

Figure 1. siPORT™ NeoFX™ Transfection Agent Effectively Delivers siRNA into Multiple Cell Types. Silencer® GAPDH siRNA or Negative Control #1 siRNA (10 nM) was transfected into the indicated cell types with siPORT NeoFX Transfection Agent. Shown are the remaining mRNA levels, as measured by real-time PCR 48 hr after transfection, relative to negative control siRNA-transfected cells.

Figure 2. Efficient Transfection with Low siRNA Concentrations. HeLa cells were trypsinized and resuspended in growth media at a concentration of 4x10 4 cells/mL. Transfection complexes were prepared using chemically synthesized GAPDH siRNA (0.03 nM to 10 nM, see graph above) or negative control siRNA and 2 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. Remaining GAPDH mRNA is shown relative to negative control siRNA-transfected cells, using 18S rRNA as an internal control.