from my experience, digestion of dsRNA with gstRnaseIII (the pnas paper) is difficult to control, it seems the 21bp is not the end product of the RNase. the power cut enzyme seems much more easy to use (http://www.econferences.de/new-tools-for-efficient-production-of-esirna-and-evaluation-of-their-functionality/), the 21bp is the end product of the recation. so, at the end of the digestion, simply desalt the reaction or use it directly! not use this enzyme

Thanks for your answer. I was wondering 2 things....you said use it directly? surely you need to purify the esiRNA molecules to get rid of all the contaminants? And at the end of your answer you say: 'not use this enzyme'. what do you mean with that?

I mean, I have not use the powecut nuclease myself. sorry for the ambiguity!

in the nature methods paper they purified the esi rna with q sepharose and isoprop precipiatation.

alternatively, you can use qiaexpress (maybe add some carrier at the precipitation step if you experience low yield)

1. Zhu, C. et al. Functional Analysis of Human Microtubule-based Motor Proteins, the Kinesins and Dyneins, in Mitosis/Cytokinesis Using RNA Interference. Mol. Biol. Cell 16, 3187-3199 (2005).

In brief, 400 base pairs 3' UTR DNA fragments of human kinesins and DHC were amplified by PCR from sheared genomic DNA of normal human foreskin fibroblasts using sequence-specific 5' and 3' primers containing T7 promoter sequence at the 5' end (Supplementary Table S1). As a control, a fragment of the luciferase coding region was amplified from a luciferase cDNA (Supplementary Table S1). The PCR products were subjected to in vitro transcription to produce dsRNAs using an in vitro T7 transcription kit (Ambion, Austin, TX). dsRNAs were then treated with DNase I, precipitated with 7.5 M LiCl, washed with 70% (vol/vol) ethanol, dried, and resuspended in water at 1 µg/µl. dsRNAs, 50 µg, were digested with 2.5 µg of GST-RNase III at 37°C for 3 h. The reactions were stopped by addition of 20 mM EDTA, and products were purified through QIAquick spin columns (Qiagen, Chatsworth, CA). esiRNAs were precipitated by ethanol and dissolved in water. The concentrations of esiRNAs were determined by measuring absorbance at UV260. The quality of esiRNAs was also evaluated by electrophoresis on 4% low-melting agarose gels.

Hi Mike,

as for Dicer vs. RNAseIII: In general RNAseIII is less sequence and fragment size specific. While this will leave you at the end with a pool of dsRNAs that are not all exactly 21bp, it allows you to generate a more complex pool of different "siRNAs". As one major advantage of esiRNA is the dilution of potential off-target effects (compared to a single or a combination of few siRNAs), a higher complexity is desirable. In addition, a lower sequence/size bias in how RNAseIII cuts should make the digestion more reproducible/stable between different sequences. This is something that matters in particular when you produce larger amounts of samples in parallel. (e.g. 96 well plates). This last comment however is rather a speculation as I have not used Dicer (or the above advertised Power Cut version) experimentally myself.

As for column vs. precipitation: A column is highly advised here (we use Q-sepharose) for two reasons. (1) In case your digestion product still contains some template or larger, not fully digested by-products you would precipitate those as well. Now, the reason why you enzymatically digest the dsRNA in vitro before transfection is that larger dsRNA molecules induce a very strong interferon response in human cells, which leads to cell death. Obviously, you don't want this. The column provides you with the necessary size selection around your target esiRNA size. (2) On the other end of the size spectrum you will also get rid of free nucleotides (and tiny digestion by-products) as well. This makes your sample both more pure and avoids (a potentially biased) contribution of free nucleotides to your NanoDrop measurements.

Best,

Dennis