History of RNAi
In 1990, researchers noticed for the first time that RNA could potentially suppress gene expression in plants. The underlying mechanism of this “erratic and reversible” gene suppression was not clear. It wasn’t until almost a decade later when Andrew Fire and Craig Mello showed in the worm Caenorhabditis elegans that double-stranded RNA (dsRNA), but not single-stranded RNA (ssRNA) was involved in gene silencing. Moreover, their report established that RNA interference (RNAi) occurs in a sequence-specific manner. This finding triggered a series of studies unraveling the detailed mechanism of RNAi over the years. In 2006, Fire and Mello were awarded the Nobel Prize for Physiology or Medicine for their seminal work.
Although researchers demonstrated RNAi functionality using exogenous dsRNAs, RNA interference is also triggered by the endogenously present single-stranded hairpin microRNAs (miRNA) in cells, small interfering RNAs (siRNA), or short hairpin RNAs (shRNA).
The primary function of natural RNA interference is to regulate gene expression. In some cases, RNAi can also confer resistance to virus or other pathogen infection.
The dsRNA that enters the cell (or the existing pre-miRNA in cells) is cleaved into smaller RNA fragments of around 21 nucleotides in length by an endonuclease, Dicer. These RNAs associate with the RNA-induced silencing complex (RISC), the antisense strand is separated from the sense strand, and are targeted to their complementary mRNA.
Following association of the siRNA or miRNA with their target, argonaute, a protein from the RISC complex, cleaves the mRNA and inhibits expression of the protein that it encodes. If the sequences of siRNA or miRNA do not perfectly match the sequence of the mRNA, then the mRNA is not cleaved, but translation is still stalled, as the RISC complex physically blocks the mRNA.
RNAi Experimental Workflow
The first step in RNAi experiments is designing the siRNAs or miRNAs. Ideally, highly specific siRNAs that target only the intended genes are desired.
Once the specific siRNAs or miRNAs are designed, they can be introduced into cells using plasmid vectors, synthetic siRNA, PCR products, or in vitro transcribed siRNAs. One advantage of RNAi is that animal cells naturally possess the endogenous machinery (Dicer and RISC) essential for the process. Therefore, few components need to be delivered inside the cells, making the experimental process relatively easy to execute.
In the last step, the efficiency of gene silencing is generally determined by measuring the mRNA transcript levels using methods such as quantitative RT-PCR measuring protein levels using immunoblotting or immunofluorescence experiments, or by monitoring obvious phenotypic changes.
The experimental workflow of RNAi experiments. The protocol for RNAi mediated gene silencing involves designing siRNAs, delivering them into cells, and analyzing the knockdown efficiency.