Much of this article was taken from Ambion's newly available RNA Interference Research Guide, which provides background information on RNAi, guidelines for optimizing siRNA delivery, recommendations for controlling siRNA experiments, an overview of siRNA screening, and detailed descriptions of the necessary tools required from start to finish for successful RNAi experiments. Request a free RNA Interference Research Guide today.

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 nearly all metazoan 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 experiments, has driven its incredibly rapid adoption by the research community.

Reagents Needed for RNAi Experiments

The required reagents for RNAi experiments are really quite simple. You need:

  1. A dsRNA (i.e., siRNA or long dsRNA) that is completely complementary to the gene transcript(s) you wish to target by RNAi
  2. A means to deliver that dsRNA to cells
  3. Proper controls
  4. A way to detect the biological effect of reducing target gene expression (i.e., an assay).

siRNA Design and Synthesis for Mammalian RNAi Experiments

For simplicity, we will confine our discussion to short term experiments in mammalian cell lines where RNAi is typically induced using short interfering RNAs (siRNAs) complementary to a desired target mRNA (Figure 1; for a more complete discussion of the mechanism and history of RNAi, see siRNAs are generally 21 bp double-stranded RNA molecules with dinucleotide 3' overhangs, and are generated intracellularly in the RNAi pathway when the nuclease Dicer cleaves long dsRNA. In mammalian cells, siRNAs have been elevated from their usual role as an RNAi intermediary to become the primary RNAi trigger used by researchers, since the long dsRNA that successfully induces RNAi in Caenorhabditis elegans and Drosophila induces a potent antiviral response in mammalian cells. siRNAs used to induce RNAi in mammalian systems are most often synthesized chemically by RNA oligonucleotide manufacturers such as Ambion, however they can also be expressed as short hairpin RNAs from a DNA construct.

Figure 1. Three Ways to Trigger the RNAi Pathway. (1) In non-mammalian systems, the RNAi pathway commences when double-stranded RNA (dsRNA; usually longer than 30 bp) is introduced into cells. In mammalian systems, RNAi can be triggered by synthetic short interfering RNA (siRNA) molecules (2) or by DNA based expression vectors designed to express short hairpin RNA (shRNA) molecules (3). In each case, gene silencing results from destruction of mRNA that is complementary to the input siRNA (2) or the siRNA molecules created by Dicer cleavage of longer dsRNA (1) or shRNA (3) molecules. See text for additional details. Dicer=cytoplasmic nuclease; RISC=RNA-induced silencing complex; mRNA=messenger RNA.

A few companies have developed complex "intelligent algorithms" that have proven to be effective at designing efficacious siRNAs. Ambion uses one of the most broadly validated intelligent algorithms to provide guaranteed-to-silence siRNAs for all human, mouse, and rat genes (see [1] for more details about this algorithm). These ready-to-use, chemically synthesized siRNAs are available individually as Silencer® Pre-designed siRNAs or Silencer® Validated siRNAs, and in genome-wide, functional class-focused, and custom sets as Silencer® siRNA Libraries and Silencer® CellReady™ siRNA Libraries. Individual siRNAs allow detailed analysis of an individual gene's role in one or more pathways, whereas siRNA libraries, or sets of siRNAs targeting a pre-defined or custom set of genes, enable large scale screening experiments to correlate genes with cellular function.

Delivery of siRNAs

Once you have an siRNA, you need a means to deliver it, and the siRNA delivery conditions need to be tested and often optimized for each cell type being used. siRNAs can be introduced directly into cells by transfection or electroporation. For many immortalized cell lines, transfection with a lipid- or amine-based reagent is the preferred option. Delivery into primary cells and suspension cells, however, often requires electroporation using a specialized, gentle-on-cells buffer, such as siPORT™ siRNA Electroporation Buffer, and optimized pulsing conditions. Such conditions generally result in very efficient siRNA delivery without compromising cell viability.

Controls for siRNA Experiments

Proper controls are needed for every experiment, and RNAi experiments are no different. Good experimental design dictates that at least two functional siRNAs to the same target should be used independently to ensure that the biological effect is due to silencing of the target gene and not due to an off-target effect. A negative control that does not target any endogenous transcript is also needed to control for nonspecific effects on gene expression caused by simply transfecting any siRNA. In addition, positive control siRNAs to easy-to-assay targets are needed to optimize transfection conditions, ensure that siRNAs are efficiently delivered, and ascertain that a particular downstream assay is working. Finally, fluorescently labeled control siRNAs facilitate the monitoring of siRNA delivery efficiency. See [2] for more information.

Assay for RNAi Effect

Assays that measure gene silencing and its effects are varied and diverse. 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 run, a researcher 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 is one that monitors target mRNA levels. The simplest and most sensitive assay for siRNA validation relies on qRT-PCR to measure target transcript levels in gene specific siRNA-treated cells versus negative control siRNA-treated cells. Many researchers also wish to determine the extent of knockdown at the protein level. Western blotting, immunofluorescence and flow cytometry are typically used for this purpose. Often time course experiments are needed to find the points of maximal mRNA and protein knockdown. Finally, some researchers will wish to correlate siRNA uptake, target mRNA, and target protein levels. Isolation and detection of small RNAs requires modified, and in some cases, completely unique techniques than isolation and detection of longer RNAs.