Key factors determining variations in RNA interference efficacy mediated by different double-stranded RNA lengths in Tribolium castaneum.

Double-stranded RNA (dsRNA) length may affect RNA interference (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 haemolymph 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 haemolymph content dynamics of different dsRNAs following injection indicated higher persistence of longer dsRNAs. In addition, comparison of the in vivo and in vitro RNAi efficacy also indicated the importance of haemolymph degradation. Thus, the varied efficacy of long dsRNAs resulted from their degradation by nucleases, which varied with dsRNA length.