The RNAi Mechanism
In non-mammalian systems, introducing or expressing long double-stranded RNA (dsRNA) triggers the RNAi pathway. The cytoplasmic nuclease Dicer first cleaves the long dsRNA into 2123 bp small interfering RNAs (siRNAs), that then unwind and assemble into RNA-induced silencing complexes (RISCs) (Figure 1). The antisense siRNA strand then guides the RISC to complementary RNA molecules, and the RISC cleaves the mRNA, leading to specific gene silencing. However, since most mammalian cells mount a potent antiviral response upon introduction of dsRNA longer than 30 bp, researchers transfect cells with 2123 bp siRNAs to induce RNAi in these systems without eliciting the antiviral response.
Figure 1. Overview of RNA Interference.
Reagents Required for RNAi Experiments
The reagents required for inducing and analyzing the RNAi effect are quite simple:
A specific dsRNA that targets a
particular gene transcript to induce the RNAi pathway
An efficient dsRNA delivery system
Assays for the RNAi effect
Tools for Non-mammalian RNAi Experiments
In non-mammalian systems such as Caenorhabditis elegans and Drosophila, long dsRNA (typically >200 bp) complementary to the target transcript is used to induce RNAi. The dsRNA can be readily generated by in vitro transcription. The MEGAscript RNAi Kit was developed specifically for this purpose. dsRNA design for inducing RNAi in many non-mammalian systems is straightforwardvirtually any long dsRNA complementary to the targeted transcript will work. Delivery methods are also typically straightforward. For example, for delivery to Drosophila S2 cells, the dsRNA can be added directly to the media used to culture them. dsRNA can also be directly injected into worms or fly embryos. Most researchers use long dsRNAs previously proved to have an easily measured biological effect as a positive control, and dsRNA that does not target any transcript in the organism (e.g., dsRNA targeting luciferase) as a negative control.
Tools for Mammalian RNAi Experiments
In mammalian cultured cells, RNAi is typically induced by siRNA introduced directly or expressed as a hairpin structure from a DNA construct within the cells. Currently, there are six methods for generating siRNAs:
In vitro preparation of siRNA
1. Chemical synthesis
2. In vitro transcription
3. Digestion of long dsRNA in vitro by RNase III or Dicer
Introduction of DNA-based vectors and cassettes that express siRNAs within cells
4. Expression from a plasmid
5. Expression from a viral vector
6. Expression from a PCR product
All of these methods, except creation of siRNA populations by digestion of long dsRNA, require careful design of the siRNA to maximize silencing of the target gene while minimizing the effects on off-target genes. Ambion supports all six siRNA synthesis methods with high quality kits and reagents. See the article in a previous issue for more details about each of these techniques and to determine the one best suited to your experimental needs.
siRNAs for Transient Transfection: Chemical siRNA Synthesis
Currently, the most widespread application of RNAi involves transient transfection of cultured mammalian cells followed by a downstream assay to monitor the RNAi effect. For this application, chemical synthesis is th e preferred and most widely used method of siRNA preparation. siRNAs are easier to transfect than plasmids. More importantly, pre-designed, gene-specific siRNAs in a ready-to-use format are available, which makes this method the easiest and the most likely to succeed.
Ambion, in partnership with Cenix BioScience, provides expert designed, guaranteed-to-silence siRNAs to >34,000 human, mouse, and rat targets (>98% of all human, mouse, and rat genes in the RefSeq database). These siRNAs are available individually as Silencer Pre-designed or Validated siRNAs, and in functional class-focused sets as Silencer siRNA Libraries. Individual siRNAs allow detailed analysis of an individual genes role in one or more pathways, whereas sets of siRNAs (libraries) enable large scale screening experiments to tie genes to cellular function.
Delivery of siRNAs into Cultured Cells
Once you have an siRNA, you need a means to deliver it into your cells. For many immortalized cell lines, transfection with a lipid- or amine-based reagent is the preferred option. Ambion's siPORT Lipid and siPORT Amine Transfect ion Agents were designed expressly for this purpose. Delivery into primary cells and suspension cells, however, can be problematic, if not impossible, using standard transfection methodologies. In these cases, electroporation using a specialized, gentle-on-cells buffer and optimized pulsing conditions generally results in very efficient siRNA delivery without compromising cell viability. Ambion's siPORT siRNA Electroporation Buffer is a revolutionary new buffer that makes electroporation an ideal method for delivering siRNAs into primary and other difficult-to-transfect cell types.
Controls for siRNA Experiments
Proper controls are needed for every RNAi experiment. A negative control that does not target any endogenous transcript is needed to control for nonspecific effects on gene expression caused by introducing any siRNA. For the vast majority of experiments, Ambion scientists use and recommend Silencer Negative Control #1 siRNA. This negative control has been extensively tested and proved to have few nonspecific effects in human, mouse, and rat cells. Easy-to-assay positive controls are needed to optimize transfection conditions, ensure that siRNAs are efficiently delivered, and ascertain that a particular downstream assay is working. Since positive cont rols are used for many different aspects of an RNAi experiment, often more than one control is required. For transfection optimization experiments, Silencer GAPDH siRNA is an ideal positive control. This siRNA efficiently silences GAPDH expression and its effects can be easily monitored by qRT-PCR or other methods at the mRNA level, or by Western blot or immunofluorescence at the protein level. In fact, Ambion provides a highly specific antibody for measuring these protein level effects.
Assay for RNAi Effect
There are several assays for measuring the RNAi effect. For understanding the biological effects of knocking down a target gene, cell based assays, enzymatic assays, array analysis, and countless other tools can be used. But before those assays can be performed, one needs to confirm that the siRNA is inducing knockdown of its intended target. siRNAs exert their effects at the mRNA level. Therefore, the preferred assay for siRNA validation and for transfection optimization purposes is one that monitors target mRNA levels. The simplest, and arguably the best, assay for siRNA validation and transfection optimization relies on qRT-PCR to measure target transcript levels in gene specific siRNA treated cells versus negative control siRNA treated cells. Applied Biosystems TaqMan Gene Expression Assays, available for >41,000 human, mouse, and rat genes, are ideal for this purpose. Ambion's siRNA database provides links to individual assays matched to gene specific Silencer Pre-designed and Validated siRNAs, which makes finding both siRNAs and real-time PCR assays to your gene of interest extremely fast and easy. Additional time can be saved with the Cells-to-Signal Kit. Typically RNA isolation is performed prior to qRT-PCR. The Cells-to-Signal Kit allows you to perform qRT-PCR directly in lysates without RNA isolation. The Cells-to-Signal procedure is compatible with TaqMan Gene Expression Assays.
Although it is necessary to monitor mRNA levels to validate siRNAs, most researchers also want to determine the extent of knockdown at the protein level. Ambions PARIS Kit (Protein And RNA Isolation System) provides a simple method for isolating total RNA and protein from the same sample. RNA isolated with the kit can be used for RT-PCR, array analysis, Northern blotting, or other analytical techniques, and the protein lysate is compatible with Western blotting. Since native protein is recovered in most cases, enzymatic assays can also be performed.
The most careful of researchers correlate siRNA, target mRNA, and target protein levels. Isolat ion and detection of small RNAs, however, requires modified, and in some cases, completely different techniques than those required for longer RNAs. The new mirVana PARIS Kit not only isolates both RNA and protein from the same sample like the PARIS Kit, but also isolates small RNA species, including siRNAs. The mirVana miRNA Detection Kit provides a method to quantitate siRNA levels in cell populations, and can also be used to examine target mRNA levels.
Thus, for performing RNAi experiments in mammalian cell systems, you need a gene specific siRNA that effectively targets the gene of interest, an siRNA delivery method, an assay to detect the RNAi effect, and proper controls. Ambion provides high quality reagents and protocols for each step of the process, so you spend less time developing and validating reagents and protocols and more time answering specific biological questions of interest to you.
Cat# Product Name Size 1552 mirVana miRNA Detection Kit 100 rxns 1556 mirVana PARIS Kit up to 40 purifications 1724 Cells-to-Signal Kit 30 rxns 1921 PARIS Kit 50 purifications 4502 siPORT Amine Transfection Agent 0.4 ml 4504 siPORT Lipid Transfection Agent 0.4 ml 4605 Silencer GAPDH siRNA (Human) 5 nmol + 2 nmol Neg Control (50M) 4611 Silencer Negative Control #1 siRNA 5 nmol (50 M) 8990 siPORT siRNA Electroporation Buffer 12 x 1.5 ml