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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. The most popular application, transient transfection of Silencer Select siRNAs or 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 (see Non-vector siRNA technologies).
For cell types that are not amenable to lipid-mediated transfection, viral vectors are often employed (see Vector-mediated RNAi). Adenoviral vectors work well for transient delivery in many cell types. However, when stable RNAi expression is desired, or for difficult cell lines, such as nondividing cells, lentiviral vectors are the best delivery method. Another approach for determining the most favorable RNAi delivery conditions is to use Life Technologies delivery optimization service—a scientific resource with extensive knowledge and expertise in viral vectors and non-viral delivery reagents for testing a matrix of delivery parameters.
Both siRNA and vector-based RNAi can be extremely effective at producing loss of function phenotypes. In general, most researchers choose siRNA because they can start quickly and there are no special preparations needed other than basic cell culture techniques. However, there are a number of reasons why a researcher might choose either siRNA or a vector-based RNAi.
Typically, researchers strive to achieve the highest levels of transfection efficiency possible. This objective is particularly important for RNAi applications because non-transfected 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 are primary cultures. However, these cannot be transfected adequately with commercially available cationic lipid-mediated transfection reagents. A powerful alternative is viral delivery of vectors expressing RNAi sequences. This option is recommended 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.
Cell type | Transient expression <7 days | Transient expression >7 days | Stable Expression |
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Fast-growing adherent cells (A549, HeLa) | Lipid transfection of Silencer Select 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 Silencer Select 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 | ||
Nondividing cells | Lentiviral delivery |
For transient knockdown experiments, synthetic, non-vector approaches offer significant advantages over vector-based methods for RNAi delivery. In particular, nonvector experiments are typically easier to design and perform and can result in higher levels of transient knockdown. In addition, recent improvements in RNAi design have increased the likelihood of achieving high-level knockdown after testing only a few RNAi molecules. Consequently, using synthetically generated RNA duplexes is the most popular method for conducting RNAi experiments.
Traditional RNAi methods for gene knockdown in mammalian cells involved the use of synthetic RNA duplexes consisting of two unmodified 21-mer oligonucleotides annealed together to form short/small interfering RNAs (siRNAs). Life Technologies’ Silencer Select siRNA and Stealth RNAi siRNA improve upon these traditional duplexes by using proprietary chemical modifications to ensure better RNAi results. Learn more about siRNA analysis
Analyses of miRNA function are performed using strategies that are similar to those used for protein-encoding genes. Transfecting cultured cells with miRNA mimics can help identify gain-of-function phenotypes; down-regulation or inhibition experiments using miRNA inhibitors can be conducted to identify loss-of-function phenotypes. The combination of up-regulation and down-regulation can be used to identify genes and cellular processes that are regulated by specific miRNAs. Learn more about miRNA analysis.
Due to their small size, these synthetic molecules are easier to transfect than vectors, and can be delivered using conditions identical to those used for siRNAs. In contrast to miRNA expression vectors, they can also be used in dose response studies.
Note: Pre-miR miRNA precursors are not hairpin constructs and should not be confused with pre-miRNAs.
siRNAs are easily introduced into cells with a siRNA transfection reagent. Soon after being inserted in the mammalian cell, the siRNA molecules become a part of the RNA-induced silencing complex (RISC). Guided by the antisense strand of the siRNA, RISC degrades the targeted mRNA inhibiting its translation. Assays are then performed to detect the RNAi activity. Controls are normally set up so RNAi results can be properly compared.
The success of RNAi is dependent on correct delivery of siRNA in appropriate amount at a time when it will brings about the maximum expected response. Such precision can be tricky. Off-targeting by siRNAs proves lethal and poses analytical issues at times. Researchers are looking for better ways of designing and delivering siRNA.
Transfection reagent | Payload | Transfection efficiency | Cell viability | Notes |
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Lipofectamine 3000 | Superior | Superior | Most efficient versatile reagent for the widest range of cell types including difficult-to-transfect cells. | |
Lipofectamine 2000 | High | High | High efficiency versatile reagent for a wide range of common cell types. | |
Lipofectamine RNAiMAX reagent | Superior | Superior | Most efficient reagent for siRNA/miRNA delivery. Efficient gene knock-down. | |
Neon electroporation | Maximal | Good | High-efficiency electroporation for all cell lines. |
Symbol | Explanation | Symbol | Explanation |
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Plasmid DNA for expression of protein, shRNA, and miRNA | mRNA for expression of protein | ||
Non-coding RNA for RNAi inhibition of gene expression | Co-delivery for cotransfection of RNAi vectors and siRNAs |
For cell types not amenable to lipid-mediated transfection, such as hard-to-transfect, primary, and non-dividing cells, viral vectors containing RNAi cassettes are often employed. Viral delivery can also be used to create stable cell lines with inducible RNAi or to express RNAi sequences with tissue-specific promoters. Adenoviral vectors work well for transient delivery in many cell types, while lentiviral vectors are best for stable delivery in dividing and non-dividing cells, lentiviral vectors are best. Learn more about vector-mediated RNAi
Viral system | When to use | |
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Adenoviral RNAi delivery |
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Lentiviral RNAi delivery |
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For Research Use Only. Not for use in diagnostic procedures.