Dissecting Systemic RNA Interference in the Red Flour Beetle Tribolium castaneum: Parameters Affecting the Efficiency of RNAi

Abstract

The phenomenon of RNAi, in which the introduction of dsRNA into a cell triggers the destruction of the corresponding mRNA resulting in a gene silencing effect, is conserved across a wide array of plant and animal phyla. However, the mechanism by which the dsRNA enters a cell, allowing the RNAi effect to occur throughout a multicellular organism (systemic RNAi), has only been studied extensively in certain plants and the nematode Caenorhabditis elegans. In recent years, RNAi has become a popular reverse genetic technique for gene silencing in many organisms. Although many RNAi techniques in non-traditional model organisms rely on the systemic nature of RNAi, little has been done to analyze the parameters required to obtain a robust systemic RNAi response. The data provided here show that the concentration and length of dsRNA have profound effects on the efficacy of the RNAi response both in regard to initial efficiency and duration of the effect in Tribolium castaneum. In addition, our analyses using a series of short dsRNAs and chimeric dsRNA provide evidence that dsRNA cellular uptake (and not the RNAi response itself) is the major step affected by dsRNA size in Tribolium. We also demonstrate that competitive inhibition of dsRNA can occur when multiple dsRNAs are injected together, influencing the effectiveness of RNAi. These data provide specific information essential to the design and implementation of RNAi based studies, and may provide insight into the molecular basis of the systemic RNAi response in insects.

Larval RNA Interference in the Red Flour Beetle, <em>Tribolium castaneum</em>

“…When EYFP dsRNA is injected in the last larval stage, a significant reduction in EYFP signal in the future wing primordia can be observed as early as one day post injection (Figure 3A). The knockdown of EYFP is complete two days after injection of the EYFP dsRNA ( Figure 3B) and this knockdown persists through the pupal stage (Figures 3C and 3D) and throughout the life of the beetle if the dsRNA concentration is high enough (such as 1 μg/μl) 7 . EYFP dsRNA can also be used as a negative control as it is a non-endogenous gene sequence and should not cause morphological or physiological disruptions (Figures 3A-3D).…”

“…Injection of dsRNA targeting EYFP can serve as a good positive control when using the pu-11 strain 5,7 . When EYFP dsRNA is injected in the last larval stage, a significant reduction in EYFP signal in the future wing primordia can be observed as early as one day post injection (Figure 3A).…”

“…The length of dsRNA molecules affects the efficiency of the systemic RNAi response, with a longer dsRNA being more efficient to trigger RNAi 7,14,15 (though the longer limit of dsRNA is currently unknown). The dsRNA length needs to be longer than 50 bp to induce effective RNAi in T. castaneum 7 .…”

“…The dsRNA length needs to be longer than 50 bp to induce effective RNAi in T. castaneum 7 . dsRNA between 150 bp and 500 bp appears to be ideal for RNAi experiments.…”

“…T. castaneum show a robust systemic RNAi response, making it possible to perform RNAi at any stage through simple injection of double-stranded RNA (dsRNA) into the beetle's body cavity [4][5][6] . Simultaneous knockdown of multiple genes is also feasible in T. castaneum by injecting two or more different dsRNA molecules at the same time 7,8 . In addition, a series of hypomorphic phenotypes can be generated by reducing the concentration of injected dsRNA 8 .…”