RNA interference (RNAi) is a natural mechanism in which small RNA molecules silence gene expression by targeting complementary mRNA for degradation or translational repression. It is one of the most powerful tools for functional genomics.
Mechanism
The RNAi pathway begins with double-stranded RNA (dsRNA) that is cleaved by the enzyme Dicer into 21–23 nucleotide small interfering RNAs (siRNAs). These siRNAs are loaded into the RNA-induced silencing complex (RISC), where the guide strand is retained and the passenger strand is discarded. The guide strand pairs with complementary mRNA, and Argonaute-2 (Ago2), the catalytic component of RISC, cleaves the mRNA, preventing translation.
Endogenous microRNAs (miRNAs) follow a similar pathway but originate from hairpin precursors (pri-miRNA → pre-miRNA → mature miRNA). Most miRNAs bind with imperfect complementarity to the 3′ UTR of target mRNAs, causing translational repression rather than cleavage.
Experimental RNAi Tools
- Synthetic siRNA: 21 bp dsRNA duplexes with 2 nt overhangs. Transfected directly into cells. Effective for 3–7 days. The most common approach for transient knockdown.
- shRNA (short hairpin RNA): expressed from a plasmid or viral vector. The hairpin is processed by Dicer into siRNA. shRNA can be stably integrated using lentiviral vectors, enabling long-term knockdown and the creation of stable cell lines.
- miRNA mimics and inhibitors: synthetic RNAs that mimic endogenous miRNAs (mimics) or block them (antagomirs, LNA probes). Used to study specific miRNA function.
Design Considerations
Effective siRNA design requires:
- Targeting the coding region or 3′ UTR of the gene
- Avoiding off-target matches (check with BLAST)
- GC content between 35–55%
- Avoiding internal repeats and secondary structure
Multiple siRNAs per target are recommended. Include a non-targeting (scrambled) siRNA control to distinguish specific from non-specific effects. A positive control (e.g., siRNA against a housekeeping gene) confirms the transfection and RNAi machinery are working.
Limitations
- Off-target effects: siRNAs can silence unintended genes through partial complementarity, particularly in the seed region (positions 2–8).
- Interferon response: long dsRNA (>30 bp) triggers an innate immune response. Use purified synthetic siRNAs to avoid this.
- Durability: siRNA is diluted by cell division. For long-term experiments, use shRNA or multiple transfections.
- Incomplete knockdown: residual protein may still be functional. Confirm knockdown at both mRNA (qPCR) and protein (Western blot) levels.
