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Among recently developed genetic tools, RNA interference (RNAi) is one of the most effective methods used in eukaryotes. Although an RNAi-like mechanism can be invoked in Toxoplasma gondii, a highly effective RNAi methodology is still elusive. Using a dual luciferase system, the ability of short (28 nucleotides) and long dsRNA (~600 nucleotides) as RNA effectors controlling gene expression in T. gondii was evaluated. An effective down regulation can be observed when ≤ 1 microgram of short dsRNAs were used in the down regulation of Renilla luciferase. We have detected that short dsRNAs are better gene modulators than long dsRNA. Most importantly, due to its high sensitivity and reproducibility, the dual luciferase reporter system reported here offers an excellent tool to be used in (i) the standardization of the RNAi silencing methodology performed in different Toxoplasma laboratories, (ii) the detailed mechanistic study of the RNAi pathway, and (iii) the development of better genetic tools for the biological study of protozoan parasites.

Evaluation of the Ability of Short and Long Double-stranded RNAs to Induce Homologous Gene Silencing in the Protozoan Parasite, Toxoplasma gondii