October 15, 2005 (Vol. 25, No. 18)
Achieving Targeted Gene Silencing Quickly and Cost-effectively
RNA interference (RNAi) is a method for targeted gene silencing. It is a highly valuable and widely used technique for the study of gene function and for drug discovery research. During RNAi, double-stranded short interfering RNAs (siRNAs), consisting of approximately 21 nucleotides with 2-nucleotide 3′-end overhangs, are introduced into cells. This results in degradation of mRNA which is complementary to the siRNA, preventing gene expression.
There are a variety of strategies for RNAi, which include the transfection of chemically synthesized siRNA, of in vitro processed siRNA, or of short hairpin RNA (shRNA)-expressing vectors. The easiest and fastest method is the transfection of chemically synthesized siRNA.
Successful RNAi experiments require the optimization of a number of different factors, including siRNA design, siRNA delivery into cells, and methods for downstream confirmation of gene knockdown. The range of RNAi solutions from Qiagen (www.qiagen.com) covers the entire RNAi workflow.
Generated using up-to-date technology and highly innovative bioinformatics, these solutions allow RNAi experiments to be performed rapidly without the need for tedious and time-consuming optimization and testing. This is advantageous both for researchers starting out in the field of RNAi and those engaged in high throughput RNAi screening projects.
siRNA design is critical for successful gene knockdown. Effective siRNA design depends on multiple factors relating to target mRNA sequence and RNAi mechanisms, and it is advisable to use an algorithm rather than selecting target sequences manually. The HiPerformance siRNA design algorithm used at Qiagen has been developed by Novartis for accurate prediction of highly potent siRNA sequences. It is a sophisticated algorithm that has extracted information about optimal design from a large set of data (3300 siRNAs for 33 genes).
siRNA specificity is also important to avoid off-target effects caused by partial homology to an unintended target. A proprietary homology analysis tool, which efficiently detects short regions of homology within an siRNA sequence, and an up-to-date, internally curated, nonredundant sequence database are used during the siRNA design process.
Using the HiPerformance algorithm and stringent homology analysis, Qiagen has designed siRNAs for every human, mouse, and rat gene. These HP GenomeWide siRNAs are available at the GeneGlobe Web portal (Figure 1; www.qiagen.com/ GeneGlobe). In addition, thousands of siRNAs targeting human genes have been tested for functionality in multiple controlled experiments. These are called HP Validated siRNAs and provide 70% knockdown. HP Validated siRNAs are provided with useful information about the cell line and transfection conditions used for validation, giving the researcher a head start in RNAi experiments.
For high throughput studies, a variety of siRNA sets are available. These range from a set for the whole human genome to sets targeting specific gene families, such as kinases and phosphatases, or custom sets. High throughput RNAi screening is becoming a widely used technique, particularly in the field of drug discovery, because of its relative ease of use and the large amount of data that can be generated.
siRNA must be delivered into as high a percentage of the cells as possible to ensure high target gene knockdown. Low transfection efficiencies lead to reduced target gene knockdown, reduced phenotypic effects, and lower reproducibility. Cells must be in optimal physiological condition and the transfection parameters (e.g., amounts of siRNA and reagent used) should be optimized.
siRNA transfection is usually carried out using a transfection reagent. It is important that the transfection reagent allows highly efficient transfection without cytotoxicity. This is especially critical for sensitive cell types such as primary cells.
In addition, it should be possible to achieve efficient transfection and knockdown with low siRNA concentrations (Figure 2). Recent studies have indicated that transfection of siRNA can result in off-target effects, in which siRNAs affect the expression of nonhomologous or partially homologous gene targets. These effects may be caused by siRNA targeting mRNA with close homology to the target mRNA, by siRNAs functioning like micro RNAs (miRNAs), or by a cellular response to siRNA toxicity.
Research suggests that off-target effects, which may produce misleading results in RNAi experiments, can be largely avoided by using low siRNA concentrations. HiPerFect Transfection Reagent allows transfection with low cytotoxicity and enables effective siRNA uptake and release of siRNA inside cells, which results in efficient transfection and knockdown using low siRNA concentrations (in some cases, less than 1 nM siRNA).
For high throughput studies, reverse transfection procedures are often used because they offer increased flexibility and speed. In reverse transfection, siRNAs are spotted into plates or onto glass slides, transfection reagent is added, and complexes are formed. Then cells are added to the siRNA-reagent complexes. Cells are usually seeded and transfected on the same day, making the procedure more rapid than traditional procedures where cells are seeded the day before transfection. A reverse transfection protocol can be found at www.qiagen.com/goto/HiPerFect.
Control experiments are vital to ensure the correct interpretation of results. Positive control siRNAs can be siRNAs that are known to result in high gene knockdown or siRNAs that effectively knock down a target gene, resulting in a particular phenotypic effect. They are used to test a transfection system or a phenotypic assay and can be routinely transfected to ensure that optimal conditions are maintained. HP Validated siRNAs are ideal for use as positive controls as they have already been shown to provide high knockdown in multiple experiments.
Negative control siRNAs can be siRNAs that are homologous to a gene that is not present in the cells under study (e.g., siRNA targeting green fluorescent protein) or siRNAs that have no homology to any known mammalian gene. Negative control siRNAs allow nonspecific effects to be monitored, and they should be routinely transfected in RNAi experiments.
siRNAs that are labeled with fluorescent dyes can be used as a control for transfection efficiency, as the number of transfected cells can be determined by fluorescence microscopy. siRNAs can be labeled with a range of dyes, including fluorescein, rhodamine, and Alexa Fluor dyes (Molecular Probes). Alexa Fluor dyes are highly photostable and bright and more persistent in cells than other traditionally used dyes.
Phenotypic effects observed after gene knockdown should be confirmed with one or more additional siRNAs targeted to different areas of the mRNA. Multiple HP GenomeWide siRNAs are available for every human, mouse, and rat gene allowing confirmation of results.
Downstream Confirmation of Gene Knockdown
Validation of gene knockdown is essential to confirm the connection between knockdown and phenotype. Quantitative, real-time RT-PCR analysis is widely used for validation of knockdown at the mRNA level.
High-quality template is essential for accurate real-time RT-PCR analysis. The FastLane Cell cDNA Kit allows high-speed cDNA preparation directly from cultured cells in four steps and less than 45 minutes. The procedure includes a step that eliminates contaminating genomic DNA, which could otherwise result in inaccurate quantification.
cDNA synthesis is the first step in two-step RT-PCR and is followed by PCR with gene-specific primers. QuantiTect Primer Assays are bioinformatically validated primer sets, which provide an economical option for specific and sensitive real-time RT-PCR. They are used in combination with QuantiTect SYBR Green Kits for detection of double-stranded DNA using SYBR Green (Molecular Probes; Figures 3 and 4).
Functional Genomics and Drug Discovery Using RNAi
RNAi experiments may involve analysis of a single gene or a small group of genes to determine gene function or elucidate biological pathways. Alternatively, whole human genome RNAi screens can be used to discover new potential drug targets. Phenotypic analysis could involve a simple cell-based assay, such as analysis of apoptosis, or a more sophisticated assay that looks at changes in the subcellular distribution of a fluorescently labeled protein.
Whatever the purpose of the RNAi experiment, each stage of the workflow, from siRNA design and delivery to downstream analysis, must be optimized to ensure the success of the experiment. The vast range of possibilities for RNAi experiments and its amenability for both low and high throughput formats will ensure that RNAi remains the technology of choice for functional genomics and drug discovery.