siLentFect™ Lipid Reagent for RNAi

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Overview

Cytotoxicity and Viability

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.

Optimal Delivery Tips

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

Culture Vessel Size
Volume of Plating Medium
siRNA Concentration
Volume of Serum-Free Medium
siLentFect Reagent
96-well
0.1 ml
5–20 nM
20 μl
0.05–0.4 μl
24-well
0.5 ml*
5–20 nM
50 μl
0.25–2.0 μl
12-well
1.0 ml*
5–20 nM
100 μl
0.5–4.0 μl
6-well/35 mm
2.5 ml*
5–20 nM
250 μl
1.0–5.0 μl
60 mm
5.0 ml*
5–20 nM
500 μl
2.5–10 μl
100 mm
10.0 ml*
5–20 nM
1.0 ml
5.0–20 μl

* Carefully aspirate medium 15–60 min prior to transfection and add one-half volume of medium.

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.

Co-Transfection Performance

Effective Silencing in Co-Transfections

Co-delivery of plasmid containing the gene along with the siRNA is a common way to optimize siRNA delivery and to validate siRNA design.  This chart shows that siLentFect™ lipid reagent is very effective for co-transfection of a plasmid and siRNA, while maintaining cell viability.

Co-transfection of plasmid DNA and siRNA. MCF-7 cells were transiently transfected in 24-well plates with different volumes of siLentFect lipid reagent, 0.5 µg of a luciferase reporter gene expression vector, and 10 nM of either a 21-mer anti-luciferase siRNA ( ) or a nonspecific siRNA control ( ). siLentFect was allowed to first complex with the siRNAs and then with the plasmid. Luciferase activity was measured 24 hr after transfection. This experiment shows that siLentFect reagent is effective at delivering both plasmid and siRNA to cells to achieve silencing of a gene expressed by a co-transfected plasmid.

Co-Transfection Tips

  • The procedure should be identical to that for delivery of siRNA alone except also add the plasmid. When using siLentFect, no additional lipid is required for a co-delivery experiment versus a single transfection
  • We have found that diluting the plasmid and siRNA separately in culture media, adding the DNA to siLentFect, followed by the addition of siRNA will result in slightly better results.  This protocol change complicates the procedure some, but improves results
  • A good practice is to always include a control with a scrambled siRNA
Transfection Protocol Library


The Transfection Protocol Online Library contains protocols obtained from the literature, developed by Bio-Rad scientists, or submitted by scientists like you. browse protocols to view our library and find your starting point.

Bio-Rad Laboratories has helped scientists deliver molecules into cells for over 35 years. Instruments, including the Gene Pulser® series of electroporators, the MicroPulser electroporator, and the Helios® gene gun and PDS-1000/He biolistic systems, have been used successfully to transfect prokaryotic and eukaryotic cells, including plant and animal cells. These transfection instruments are cited extensively in the literature. Our chemically mediated methods include lipid-based transfection reagents — TransFectin lipid reagent for delivery of plasmid DNA and siLentFect lipid reagent for siRNA delivery.

siLentFect reagent is a high-efficiency cationic lipid developed for the transfection of cultured mammalian cells with short interfering RNA (siRNA). Effective delivery of the siRNA molecules is critical to the success of RNA interference (RNAi) applications. siLentFect has high affinity for the small siRNA molecules, which helps to achieve robust silencing of the target gene with low lipid volumes and low siRNA concentrations. siLentFect reagent is a powerful delivery tool that maximizes efficiency while minimizing cytotoxicity at optimal delivery concentrations.

siLentFect lipid reagent is a mixture of a proprietary cationic compound and a co-lipid. The blend has been optimized for the intracellular delivery of siRNAs into cultured mammalian cells in the presence of serum at cell densities from 50 to 90%. For most cell lines, high levels of gene silencing can be obtained using amounts of siLentFect reagent and siRNA suggested for given sizes of plates/wells.

Key Application

RNAi is a powerful tool used to manipulate gene expression and allow researchers to determine gene function. RNAi is the sequence-specific gene silencing induced by double-stranded RNAs. siRNAs are double-stranded RNA molecules, typically 21–27 nucleotides in length that greatly stimulate gene-specific silencing. siLentFect reagent has been developed to produce exceptional delivery of siRNA to mammalian cells in culture.

Key Features

  • Effective — successfully transfect siRNA into a broad range of cell lines
  • Economical — low volumes of lipid required per transfection help to minimize cell stress while saving money on each transfection
  • Specific — use 5 nM or less siRNA for effective knockdown and reduce the risk of off-target effects
  • Viable — low cytotoxicity facilitates robust knockdown while maintaining culture health
  • Flexible — excellent performance at culture densities between 50 and 90% and in the presence or absence of serum-containing medium

Other Lipid Reagents

Bio-Rad offers a family of lipid transfection reagents, each of which is optimal for certain applications. siLentFect lipid reagent is an RNAi-specific lipid that is ideal for siRNA delivery. It can also be used to perform cotransfection with a small reporter-plasmid along with the siRNA molecule. TransFectin™ lipid reagent is a general-purpose lipid that is ideal for plasmid delivery.

siLentFect™ Lipid Reagent for RNAi

170-3360
0.5 ml, lipid transfection reagent, RNAi-specific lipid for siRNA delivery

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siLentFect™ Lipid Reagent for RNAi

170-3361
1.0 ml, lipid transfection reagent, RNAi-specific lipid for siRNA delivery

List Price:   $457.00
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siLentFect™ Lipid Reagent for RNAi

170-3362
5 x 1.0 ml, lipid transfection reagent, RNAi-specific lipid for siRNA delivery

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Number Description Options
3105 siLentFect Lipid Reagent Flier, Rev B Click to download
4106271 Instruction Manual, siLentFect Lipid Reagent, Rev A Click to download
5343 Transfection Reagent References, Rev C Click to download
5439 Highly Efficient Transfection of a Human Epithelial Cell Line With Chemically Synthesized siRNA Using siLentFECT Lipid Reagent, Rev A Click to download
5370 Transfection of Caco-2 Cells With siRNA Using the siLentFect Lipid Reagent, Rev A Click to download
5807 Beta-Actin Gene Silencing via siRNA and Its Effects on Protein Profiles (Poster), Rev B Click to download
5808 Novel Uses of Microarrays in Detecting Gene Silencing (Poster), Rev A Click to download
5894 Highly Efficient Reverse siRNA Transfection of Human Epithelial Lung Carcinoma A549 Cells Using siLentFect Lipid Reagent, Rev A Click to download