Key factors determining variations in RNAi efficacy mediated by different dsRNA lengths in Tribolium castaneum.

dsRNA length may affect RNAi efficacy. Herein, variation in RNAi efficacy associated with dsRNA molecular length was confirmed via comparison of knockdown results following dsRNA injection into Tribolium castaneum. Through in vitro experiments with T. castaneum midgut, dsRNA accumulation in the midgut, degradation by midgut homogenates, and persistence in hemolymph after injection were tested to determine the causes of RNAi efficacy variation. The comparative efficacies of dsRNAs were 480 bp ≈ 240 bp>120 bp>60 bp>21 bp. The combined midgut dsRNA accumulation and midgut homogenate-induced degradation analyses suggested cellular uptake to be the key barrier for 21 bp dsRNA functioning, but was likely not the main determinant of the variation in longer dsRNAs (≥ 60 bp) bioactivity. in vitro RNAi experiment with T. castaneum midgut showed that long dsRNAs all significantly depleted the expression of corresponding genes, suggesting little variation in intracellular RNAi machinery's affinity for different dsRNA lengths. in vivo hemolymph content dynamics of different dsRNAs following injection indicated higher persistence of longer dsRNAs. Besides, comparison of the in vivo and in vitro RNAi efficacy also indicated the importance of hemolymph degradation. Thus, the varied efficacy of long dsRNAs resulted from their degradation by nucleases, which varied with dsRNA length. This article is protected by copyright. All rights reserved.