There has been limited success in using short interference RNA (siRNA) or short hairpin RNA (shRNA) to trigger RNA interference (RNAi) and knockdown genes in insect systems. Instead, long double stranded RNA (dsRNA) is typically used. Several reasons for why siRNA is not effective in insect systems are proposed, including length dependent dsRNA uptake in cells and position effect of siRNAs. Here, we compared the potency of si/shRNA and dsRNA in CPB cells. In CPB cells, si/shRNA was unable to trigger RNAi. With the help of transfection reagent, si/shRNA was able to trigger RNAi in CPB cells. Confocal observations of Cy3 labeled-si/shRNA cellular uptake revealed reduced uptake of si/shRNA compared to dsRNA. 32P labeled-si/shRNA was processed successfully into smaller (17 – 21bp) siRNA in CPB cells, implicating that Dicer processing efficiency is not the reasons for low potency of si/shRNA. We were able to show that long dsRNA fused si/shIAP effectively trigger RNAi in CPB cells, suggesting that the length of si/shIAP is critical parameter. Our results show that CPB cells are unable to uptake short length si/shRNA and this leads to ineffective RNAi response.
