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.

Figure 1. The effect of dsRNA size on RNAi knockdown efficiency.

(A) Location of EGFP dsRNAs relative to the EGFP gene. Green: the full-length EGFP gene (minus noncoding portion). Red: long dsRNA (520 bp). Purple: intermediate dsRNA (69 bp). Blue: short dsRNA (30/31 bp). (B) Uninjected pu11 Tribolium larvae. (C-H) pu11 Tribolium larvae injected with EGFP dsRNA or siRNA. (C) 520 bp dsRNA. (D) 69 bp dsRNA. (E) 21 bp siRNA. (F) 31 bp (142–172 bp) dsRNA. (G) Two 31 bp dsRNAs. (H) Eight ∼30 bp dsRNAs. (I,J) pu11 Tribolium larvae injected as embryos with EGFP dsRNA. (I) Top larvae; uninjected, Middle larvae; injected with eight ∼30 bp dsRNAs, Bottom larvae; injected with 520 bp dsRNA. (J) Light microscopy image of (I).

Figure 2. The effect of dsRNA concentration on RNAi knockdown efficiency.

(A) Weight dilution series of 520 bp EGFP dsRNA injected into pu11 Tribolium larvae. (1) 0.01 µg/µL. (2) 0.001 µg/µL. (3) 0.0001 µg/µL. (4) 0.00001 µg/µL. (B) Weight dilution series of 69 bp EGFP dsRNA injected into pu11 Tribolium larvae. (1) 0.01 µg/µL. (2) 0.001 µg/µL. (3) 0.0001 µg/µL. (4) 0.00001 µg/µL. (C) Molar dilution series of 520 bp EGFP dsRNA injected into pu11 Tribolium larvae. (1) ∼0.07 µg/µL. (2) ∼0.007 µg/µL. (3) ∼0.0007 µg/µL (4) ∼0.00007 µg/µL. (D) Weight dilution series of 520 bp EGFP dsRNA injected into a tubulin EGFP Tribolium larvae. (1) 0.01 µg/µL. (2) 0.001 µg/µL. (3) 0.0001 µg/µL. (4) 0.00001 µg/µL.

Figure 3. Reduction of EGFP mRNA by various sizes and concentrations of EGFP dsRNA.

(A) Location of EGFP dsRNAs and qPCR amplicon relative to the EGFP gene. (B) Reduction of EGFP mRNA induced by various sizes of EGFP dsRNA. (C) Reduction of EGFP mRNA induced by various concentrations of EGFP 480 bp dsRNA. (D) Reduction of EGFP mRNA induced by various concentrations of EGFP 60 bp dsRNA. Arrows in C and D indicate a potential threshold concentration for inducing efficient RNAi.

Figure 4. The effect of dsRNA size and concentration on the duration of the RNAi effect.

(A) Line graph showing the percentage of individuals expressing EGFP after injection of EGFP dsRNA at three different conditions. (B) pu11 Tribolium injected with 520 bp EGFP dsRNA at a concentration of 1 µg/µL. (C) pu11 Tribolium injected with 520 bp EGFP dsRNA at a concentration of 0.01 µg/µL. (D) pu11 Tribolium injected with 69 bp EGFP dsRNA at a concentration of 0.01 µg/µL. Asterisk indicates the first day EGFP expression was detected in the adult eye.

Figure 5. The effect of dsRNA competition on RNAi knockdown efficiency.

(A) Competitor dsRNA injected alone. (1) DsRed dsRNA. (2) Ubx dsRNA. (B) Competitor and reporter dsRNA co-injected at a 10 to 1 ratio. (1) DsRed dsRNA injected at 1 µg/µL (competitor). EGFP dsRNA injected at 0.1 µg/µL (reporter). (2) Ubx dsRNA injected at 1 µg/µL (competitor). EGFP dsRNA injected at 0.1 µg/µL (reporter). (C) Competitor and reporter dsRNA co-injected at a 100 to 1 ratio. (1) DsRed dsRNA injected at 1 µg/µL (competitor). EGFP dsRNA injected at 0.01 µg/µL (reporter). (2) Ubx dsRNA injected at 1 µg/µL (competitor). EGFP dsRNA injected at 0.01 µg/µL (reporter). (D) Competitor and reporter dsRNA injected sequentially at a 100 to 1 ratio. (1) DsRed dsRNA injected at 1 µg/µL (competitor). EGFP dsRNA injected at 0.01 µg/µL (reporter). (2) Ubx dsRNA injected at 1 µg/µL (competitor). EGFP dsRNA injected at 0.01 µg/µL (reporter). (E) DsRed dsRNA or DsRed dsDNA is used as the competitor nucleic acid. EGFP dsRNA is co-injected as the reporter. The ratio of competitor to reporter is 100 to 1. (1) DsRed dsRNA injected at 1 µg/µL. EGFP dsRNA injected at 0.01 µg/µL. (2) DsRed dsDNA injected at 1 µg/µL. EGFP dsRNA injected at 0.01 µg/µL.