siLentFect Lipid Reagent for RNAi

Cytotoxicity

It is very important that RNAi experiments use a delivery method that results in very little cytotoxicity. Cytotoxicity can impact results in a few different ways:

• "Silencing bias" due to toxicity: If cells are exposed to toxic levels of a substance, they may lyse and die. This can have a big impact on end-point data. Unless proper controls are run, what looks like good silencing could in fact be cell death. Further, cell death reduces the amount of total signal from a culture, making data less robust
• Translational arrest: Cells can undergo translational arrest, meaning that they will slow metabolism such that very few proteins are being translated. This can happen if cells are exposed to toxic concentrations of a substance (or long dsRNA)

Toxicity is best evaluated by practiced tissue culturists using visual cues. Morphological changes, changes in growth, and gaps in culture density are indications that a culture has been exposed to toxic concentrations of a substance. The following images provide examples of cultures exposed to low, moderate, and high concentrations of a toxic substance. Different levels of toxicity can be defined by visual analysis of cells in culture. As cells are exposed to higher concentrations of a toxic substance, their morphology changes from A, oblong-shaped cells to C, rounded shapes. As the cells continue to round, they eventually detach from the plate or lyse and die.

High Efficiency of siRNA Delivery with Low Toxicity

siLentFect™ is very effective at cytoplasmic delivery of siRNA. Very low volumes of lipid are required per transfection, reducing the cost of each knockdown experiment.

Two characteristics of siLentFect are illustrated below. First, high efficiency is shown by using very low concentrations of siLentFect to achieve sufficient knockdown (ideal range being 0.75 to 2 µl in a 24-well plate). Second, low toxicity is shown by observing the β-galactosidase expression of the nonspecific control siRNA (shown in orange), which was consistent with the untransfected control. With increasing concentrations of siLentFect, culture health is maintained, indicating that siLentFect is minimally toxic at optimal concentrations.

β-galactosidase knockdown using siLentFect and anti-β-gal siRNA. Stable CHO-lacZ cells were produced using the Gene Pulser Xcell™ system and seeded into 24-well plates. Cells were then transfected with 10 nM anti-β-gal siRNA (blue) or a nonspecific control siRNA (orange) using increasing amounts of siLentFect. After 24 hr, cells were assayed for β-gal activity.

Note: The β-galactosidase is a good model because visual x-gal staining for qualitative and ONPG assays for quantitative analyses can be performed. However, β-galactosidase protein is very stable, so ONPG assays for knockdown 24 hr posttransfection are not as representative of reductions in transcript amounts. Knockdown results are biased by the presence of protein that was expressed prior to siRNA delivery, but remains in the cell at the time of assay. This explains the importance of prior characterization of protein stability when using qPCR for RNAi analysis. Knockdown may be 99% of transcript after 24 hr, but persistent protein stability may alter the phenotypic assay results.

Silencing Bias Due to Toxicity

Exposure to toxic levels of a substance can cause cells to lyse and die. This can have a significant impact on evaluating end-point data. In the absence of proper controls, a decrease in expression may appear to represent good silencing but could in fact be due to cell death. Further, cell death reduces the amount of total signal from a culture, making data less robust.

This chart shows the effects of increasing concentrations of two lipids, siLentFect and another lipid (Lipid X). The "anti-luc" lipid traces show knockdown of luciferase in CHO cells stably transfected to express luciferase. However, the control (no knockdown) for Lipid X shows that with increasing concentrations of lipid, the expression of luciferase decreases. This is likely due to cell stress or cell death. siLentFect displays minimal toxicity as shown by only slight reduction in expression for the control sample.

Impact of Toxicity on Optimization

The toxicity of the lipid can influence the amount of optimization that can be performed. If the lipid has a small window of functionality before causing toxicity effects, then the protocol is limited. siLentFect has a broad functional range, in part due to its low toxicity, which allows for greater protocol optimization to accommodate a variety of cell lines. The charts below illustrate the functional range of siLentFect as compared to another available lipid.

COS-7 cells were seeded into 24-well plates and transfected with 0.5 µg of a luciferase reporter gene expression vector (pCMViLuc) and 10 nM of either an anti-luciferase siRNA (—) or a nonspecific siRNA control (—) using different volumes of siLentFect reagent and another lipid (Lipid X). Luciferase activity was measured 24 hr posttransfection. The "anti-luc" traces (blue) show excellent knockdown of luciferase using both lipids, however the control samples (green) show dramatically different results. The siLentFect control sample remains steady, indicating consistent cell viability and very low toxicity. In contract, the Lipid X control sample shows a significant decrease in expression, which is likely due to cell death from high toxicity.

Optimization

Determining the optimum conditions for transfection efficiency is essential to maximize gene silencing and to minimize cellular toxicity. The two most important parameters to optimize for any given culture vessel and cell density are the amount of siLentFect lipid reagent and the concentration of siRNA. If toxicity is encountered try reducing lipid amounts used in transfection. The amount of siLentFect lipid reagent and the concentration of siRNA required for maximal gene silencing can vary among different cell types.

Typically, siLentFect™ lipid reagent requires less reagent than other lipids for effective siRNA delivery. When working with different lipids or new cell lines, it is important to perform a dilution series of both siRNA and lipids to ensure optimal results.

Optimizing the lipid volume and siRNA concentration. A, CHO cells, stably expressing the lacZ gene, were grown in 24-well plates and transfected with an siRNA targeting lacZ. Cells were lysed and assayed for β-galactosidase activity 24 hr posttransfection. A significant reduction in expression of the lacZ gene product was observed even with low volumes of siLentFect. B, CHO cells, stably expressing the luciferase gene, were transfected in 96-well plates using 0.3 μl siLentFect and a 21-mer anti-luciferase siRNA. The addition of siRNA at concentrations >10 nM did not produce any further significant reduction in luciferase activity. These data also illustrate how siLentFect mediates efficient transfection using very low siRNA concentration and lipid volume.

Suggested Reagent Quantities for Different Sizes of Plates or Wells

Other Recommendations for Best Results

siLentFect lipid reagent has been developed to achieve consistent transfection efficiencies using a broad range of cell types with an easy-to-use protocol. Optimum transfection efficiencies are achieved by adjusting:

• Quantities of siLentFect reagent
• siRNA concentration
• Cell density at the time of transfection
• Length of exposure of cells to siLentFect-siRNA complexes

Once maximum transfection efficiency has been established, the conditions should be kept constant between experiments for any particular cell line.

• Invert the tube of siLentFect to mix contents before using
• Use sterile polystyrene plasticware (for example, 12 x 75 mm tubes or multi-well trays) to prepare the siRNA solutions and lipid solutions. Polystyrene is recommended because cationic lipid-siRNA complexes may bind to polypropylene

NIH-3T3 cells transfected with fluorescently labeled siLentMer™ nonsilencing siRNA using siLentFect lipid reagent. At 24 hr posttransfection, cells were fixed and stained with Hoechst dye. A, cells were imaged using brightfield optics B, and then analyzed by fluorescent microscopy to detect nuclear staining C, and siRNA intake. The visualization of the fluorescently labeled siRNA can provide an indication of the transfection efficiency.