RNAi: Size Does Matter

Everyone wants to design the dsRNA that most efficiently silences the gene of interest. But how does dsRNA length correlate with silencing efficiency? Ambion scientists addressed this question in the
following experiment.

dsRNA of different sizes and directed against different regions of hrp48 and U2AF⁵⁰ cDNA (Figure 1A), were prepared using the MEGAscript^™ RNAi Kit and tested on Drosophila L2 cells. For both targeted genes, Western blot analyses showed a stronger silencing effect with dsRNA corresponding to the full length cDNA than with shorter dsRNAs (Figure 1B and D). Similar reduced activities (in comparison to that of full length dsRNAs) were obtained with shorter dsRNA specific for either the 5' or the 3' end of U2AF⁵⁰ cDNA (Figure 1B).

Interestingly, the high level of silencing induced by the longer dsRNAs could be reached with the shorter dsRNAs by increasing the concentration of the shorter dsRNAs 2-3 fold (Figure 1D). Because yields of dsRNA synthesized and purified with the MEGAscript RNAi Kit are reproducibly higher for shorter dsRNA (Figure 1C), two to five time more experiments can be performed per transcription reaction with 300 to 600 nt long dsRNA. Therefore, it may be more convenient and cost effective to perform silencing experiments with the higher concentration of shorter dsRNA.

Figure 1. Correlation Between dsRNA Size, Transcription Yield and Silencing Effect. (A) Schematic representation of the various dsRNA used in this study. (B) Western blot analysis 72 hours post treatment with 10 nM of the indicated dsRNA. (C) Yields of dsRNA synthesized and purified with the MEGAscript RNAi kit using 0.4 µg of PCR DNA templates containing T7 RNA polymerase promoters on both ends. (D) Western blot analysis 72 hr post treatment with different concentrations of the indicated dsRNA.

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