Selecting an RNAi Strategy

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.

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

• Silencer siRNAs are Ambion-designed siRNAs available for all human, mouse, and rat gene targets in the RefSeq database. These siRNAs are designed for maximum potency and specificity using a highly effective and extensively tested algorithm. Each siRNA is synthesized to the highest quality standards and is provided with full sequence information.
• Stealth RNAi siRNA molecules are chemically modified, blunt-ended, 25-mer double-stranded duplexes that are recognized by the RNA-induced silencing complex (RISC) to mediate inhibition of a target gene. Proprietary chemical modifications allow Stealth RNAi siRNA to overcome many in vivo–specific obstacles, allowing effectiveness and stability in in vivo applications.
• Silencer Select siRNAs are the best-performing siRNAs for in vitro studies, and are available in a variety of formats including preplated collections and custom libraries to simplify screening experiments. They are up to 100-fold more potent than other siRNAs (modified and unmodified), allowing a higher percentage of “on-target” phenotypes.

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.

• Ambion Pre-miR miRNA Precursors are small, chemically modified double-stranded RNA molecules that are similar, but not identical, to siRNAs, and are designed to mimic endogenous mature miRNAs.
• mirVana miRNA Mimics are small, chemically modified double-stranded RNAs that mimic endogenous miRNAs and enable miRNA functional analysis by up-regulation of miRNA activity. These molecules are more specific than their predecessors due to inactivation of the star strand by proprietary chemical modifications. mirVana miRNA Mimics are available individually or as libraries
• Ambion Anti-miR miRNA Inhibitors are chemically modified single-stranded nucleic acids designed to specifically bind to and inhibit endogenous miRNAs.
• mirVana miRNA Inhibitors are small, chemically modified single-stranded RNA molecules designed to specifically bind to and inhibit endogenous miRNA molecules and enable miRNA functional analysis by down-regulation of miRNA activity. They have the highest-potency in vitro inhibition at the lowest miRNA inhibitor concentration of available miRNA mimics. mirVana miRNA Inhibitors are available individually or as libraries.

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.

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.

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

• BLOCK-iT Adenoviral RNAi Expression System facilitates the creation and delivery of a replication-incompetent adenovirus to transiently express shRNA in most dividing or non-dividing mammalian cell types and animal models for RNAi analysis. The key advantage of the BLOCK-iT Adenoviral RNAi Expression System is Gateway® recombination technology, which simplifies the cloning and generation of an adenoviral vector, eliminating the tedious and time-consuming manipulations, screening, and multiple transformations that other adenoviral systems require.
• BLOCK-iT Lentiviral RNAi Expression System enables the creation and delivery of engineered shRNAs and miRNAs into dividing and non-dividing mammalian cells, including primary and hard-to-transfect cells. The system can be used without selection for transient RNAi analysis or, with appropriate antibiotic selection, to generate a stable cell line for long-term knockdown studies.
• BLOCK-iT Lentiviral Pol II miR RNAi Expression System combines BLOCK-iT Pol II miR RNAi and ViraPower Lentiviral technologies to facilitate the creation and stable delivery of engineered miRNAs into nondividing, primary, and hard-to-transfect cells. The Pol II promoter in the expression vector enables co-cistronic expression of multiple miRNAs, allowing knockdown of multiple targets from a single construct, a process is ideal for knockdown of more than one pathway component or splice variant, or for using knockdown to create synthetic phenotypes.
• BLOCK-iT Lentiviral Pol II miR RNAi Expression System with EmGFP provides all the components and benefits of the BLOCK-iT Lentiviral Pol II miR RNAi Expression System listed above, plus the easy expression tracking with co-cistronic EmGFP. The HiPerform version of the expression vector contains an mRNA-stabilizing sequence (WPRE) and a nuclear import sequence (cPPT) that can generate up to 5-fold higher virus titers.
• BLOCK-iT Inducible H1 Lentiviral RNAi System is a complete lentiviral system for long-term inducible or constitutive shRNA expression in any cell type. Regulation of the RNAi response via the tetracycline operator (TetO₂) sequence permits the study of changes over time and loss-of-function experiments even with essential genes, and provides an excellent control system to measure phenotypic changes during recovery of gene function.