The two common approaches for RNAi delivery are lipid-mediated transfection and viral-mediated transduction. Determining which one of these approaches to use depends on the cell type being studied and whether transient or stable knockdown is desired (Tables 1 & 2). The most popular application, transient transfection of unmodified siRNAs or modified Stealth RNAi siRNA duplexes, uses cationic lipid-based reagents because they are suitable for delivering molecules across a diverse range of commonly used cell lines. For cell types not amenable to lipid-mediated transfection, viral vectors are often employed. Adenoviral vectors work well for transient delivery in many cell types; however, for some difficult cell lines, such as non-dividing cells and for stable RNAi expression, lentiviral vectors are the best delivery method. Another approach for determining the most favorable RNAi delivery conditions is to use Invitrogen’s delivery optimization service - a scientific resource with extensive knowledge and expertise in viral vectors and nonviral delivery reagents for testing a matrix of delivery parameters.
Table 1. Recommended RNAi delivery methods
||Transient expression (<7 days)||Transient expression (>7 days)||Stable expression|
|Fast growing Adhernet cells (A549, Hela)||Lipid transfection of siRNA or Stealth RNAi siRNA||Lipid transfection of RNAi vectors or Adenoviral delivery||Lipid transfection of RNAi vectors or Lentiviral delivery|
|Fast growing suspension cells (THP-1)||Lipid transfection or electroporation of siRNA or Stealth RNAi siRNA||Lipid transfection of RNAi vectors or Adenoviral delivery||Lipid transfection or electroporation of RNAi vectors or Lentiviral delivery|
|Primary cells||Lentiviral delivery|
|Non-dividing cells||Lentiviral delivery|
Table 2. Selecting transfection reagents and viral delivery methods
|Lipofectamine RNAiMAX Transfection Reagent||
|Lipofectamine 2000 Transfection Reagent||
|Oligofectamine Transfection Reagent
|BLOCK-iT Adenoviral RNAi Expression System
|BLOCK-iT Pol II miR RNAi Lentiviral Expression System
BLOCK-iT Lentiviral RNAi Expression System
BLOCK-iT Inducible H1 Lentiviral RNAi System
Methods to achieve high transfection efficiency
Transfection efficiency describes the percentage of cells that have received the RNAi duplex or expression plasmid. Typically, researchers strive to achieve the highest levels of transfection efficiency possible. This objective is particularly important for RNAi applications because nontransfected cells will continue to express the gene targeted for knockdown, thus contributing to background expression levels.
For many disease models, the most desirable cell types to use are primary cultures. However, these cannot be transfected adequately with commercially available transfection reagents. A powerful alternative to cationic lipid-mediated transfection is viral delivery of vectors expressing RNAi sequences. This option is best for delivery to hard-to-transfect, primary, and nondividing cells. Viral delivery can also be used to create stable cell lines with inducible RNAi expression or to express RNAi sequences with tissue-specific promoters.
Importance of minimizing transfection-mediated cytotoxicity
The delivery of RNAi reagents, or the delivery method itself, can give rise to cytotoxicity in gene silencing experiments. Minimizing transfection-mediated cytotoxicity is essential for proper interpretation of the outcome of any RNAi experiment, as cytotoxic effects might be difficult to distinguish from a phenotype resulting from target gene knockdown. Cytotoxicity from the delivery method should be suspected if apparent knockdown of the target gene is seen when cells are transfected with a negative control with similar GC content to that of the target gene. The easiest way to combat the issue of delivery-related cytotoxicity is to choose a transfection reagent designed for either double-stranded RNA or plasmid-based RNAi transfections. Most often, these reagents are formulated for maximizing efficiency (to achieve high knockdown levels) while minimizing cytotoxicity. Optimization experiments via supplier or published protocols as guidelines can help to determine which concentration of transfection reagent works best for the cell line of interest. We recommend using the lowest amount of transfection reagent necessary to give the highest level of knockdown.
Significance of reducing off-target effects
As well as being a source of cytotoxicity, a suboptimal delivery reagent or excess reagent can result in apparent off-target effects. One cause of off-target effects is the up- or down-regulation of genes due to the gene delivery procedure. However, with appropriate controls, these effects can be identified and diminished. Again, using the least amount of transfection reagent that provides the best gene silencing activity is advised.
The potential exists for off-target effects due to knockdown from the siRNA duplex itself. For determining the most favorable conditions, the concentration of siRNA should be varied while the concentration of transfection reagent is held constant at the lowest concentration previously identified. The lowest siRNA concentration that gives the desired level of knockdown in RNAi experiments should be utilized. Keep in mind that the specificity of the siRNA will have an impact on potential off-target effects. The use of exceptionally specific reagents, such as modified Stealth RNAi™ siRNA duplexes, can help alleviate these concerns.
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