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RNAi: A “How To” for New Users
RNA interference, the biological mechanism by which double-stranded RNA (dsRNA) induces gene silencing by targeting complementary mRNA for degradation, is revolutionizing the way researchers study gene function. For the first time, scientists can quickly and easily reduce the expression of a particular gene in mammalian cell systems, often by 90% or greater, to analyze the effect that gene has on cellular function. The ease of the technique, as well as the wide availability of high quality kits and reagents for performing RNAi, have contributed to its rapid adoption by the research community. This article provides an overview of RNAi, the requirements for a typical RNAi experiment, and the Ambion products that simplify each step.
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 21–23 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 21–23 bp siRNAs to induce RNAi in these systems without eliciting the antiviral response.
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| 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
• Proper controls
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 straightforward—virtually 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 the 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 gene’s 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 Transfection 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 controls 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. Ambion’s 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. Isolation 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.
Summing Up
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.
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