Excerpts from RNA Interference Research Guide
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.
Reagents Needed for RNAi Experiments
- A dsRNA (i.e., siRNA or long dsRNA) that is completely complementary to the gene transcript(s) you wish to target by RNAi
- A means to deliver that dsRNA to cells
- Proper controls
- A way to detect the biological effect of reducing target gene expression (i.e., an assay).
siRNA Design and Synthesis for Mammalian RNAi Experiments
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  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.