RNAi experiments involve three major steps:
1) Production of dsRNA matching gene of interest
• RNA is synthesized in a test tube by adding phage RNA polymerases that recognize phage promoters housed in expression vectors
• T7 and T3 promoters enable transcription of individual strands of the gene when T7 or T3 polymerases are provided
• Both promoters flank the cloned gene
• Usually T7 polymerase drive expression of one strand (anisense) and T3 the complementary strand (sense)
• The two strands are annealed together to make dsRNA
2) Initiation step
• Input of dsRNA - RNAs could be injected directly into the target tissue.
C. elegans and other nematodes can uptake dsRNA when soaked in dsRNA solution or fed plasmids that make dsRNA and consequently exhibit RNAi effects. (Tabara et al., 1998) This is the special characteristic of RNAi where dsRNAs can be transported across cell boundaries and be distributed to target tissues.
• Inside the organism, the dsRNA is processed into 21-23 nucleotide siRNAs. Multiple duplexes of siRNAs are produced by successive cleaveage of a nuclease complex at the 3’ end of the dsRNA
• RNA amplification (the mechanism is still unclear. An RNase III-like activity maybe involved in the RNAi mechanism although the evidence for this hypothesis remains inconclusive)
3) Formation of RNA-induced silencing complex (RISC)
• siRNAs are incorporated into the RISC nuclease complex
• RISC targets the homologous transcript by base pairing interactions between one of the siRNA strands and the endogenous mRNA. It then cleaves the mRNA at the 3' terminus of the siRNA
• Destruction of the target mRNA silenced gene
• Phenotypic analysis - phenotypes were tested differently at different time periods according to the phenotypic class. Observations include brood size and progeny viability
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