RNA Interference (RNAi ) is widely used by researchers of the past decade in functional genomics. As one of the most important breakthroughs in molecular biology, RNAi technology allows us to directly observe specific gene silencing in eukaryotes. The phenomenon of RNAi is highly conserved among species. Since the groundbreaking discovery of RNAi in C. elegans, the model organism C. elegans has been widely used to study the mechanism of RNAi.
Extensive genetic and biochemical analysis revealed a two-step mechanism of RNAi-induced gene silencing. First, degradation of dsRNA into small interfering RNAs (siRNAs, 21~25 nt), by an RNase III-like activity. Second, the siRNAs join an RNase complex, RISC (RNA-induced silencing complex), which acts on the cognate mRNA and degrades it.
Figure 1. Mechanism of RNA interference (RNAi) - Creative Biogene
Nowadays, RNAi has been applied to systematically shut down the expression of each gene to conduct genome-wide screens in C. elegans. For RNAi, double-stranded RNA (dsRNA) can be introduced in the worms by injection, soaking or feeding.
- Microinjections. dsRNA can be injected into the intestine, gonad, or body cavity. Because the injected worms must be examined at different time points to determine the optimum screening time, microinjections are not practical for performing large-scale RNAi screens.
- Soaking. Soaking the worms in a concentrated solution of dsRNA also induces RNAi as dsRNA is ingested through the pharynx. The soaking approach is not as tedious as microinjections and also amenable to high throughput screening. However, it is expensive due to a large amount of in vitro transcribed dsRNA required for soaking the worms.
- Feeding. dsRNA transcription can be ingested by C.elegans. This procedure is performed on petri plates or in liquid culture. RNAi by feeding in liquid culture is ideal for scoring easily observable phenotypes such as sterility, lethality, slow growth and dramatic changes in GFP expression. RNAi screens by feeding are far less labor-intensive and also inexpensive and are commonly used.
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* For research use only.