My research objective is to silent a targeted gene through RNA interference (RNAi). I cloned my designed gene fragment (double stranded RNA) in L4440 vector under the control of two T7-promoters ( as shown in picture) to get a double stranded RNA (dsRNA). This cloned vector is transformed into a HT115 RNase-deficient E. coli. To isolate dsRNA I extracted total nucleic acid of HT115 RNase-deficient E. coli. This total nucleic acid was treated with DNase I to remove DNA content and some of its fraction was visualized on agarose gel. The remaining nucleic acid was treated with RNase A (Thermo Scientific, Catalog number: EN0531) to remove single stranded RNA in presence of 1M NaCl and 0.2ug/ul RNase A for 30-60 minutes at 37°C. when i visualized RNase A treated samples in agarose gel I could not observe dsRNA. RNase A treatment also digests dsRNA. If i increase concentration of NaCl even ssRNA could not be digested.
Kindly help to me purify intact dsRNA from HT115 RNase-deficient E. coli.
Thanks in anticipation..
You can try RNase I but I recommend using S1 nuclease or mung bean nuclease. They are a lot more efficient than RNase I, very specific to ssRNA and ssDNA, very stable and less prone to inhibition.
Best
Jazak Allah @ Waleed Albihlal
I have to purchase S1 nuclease and Mung bean nuclease. Can you suggest me which product/company would be better?
I buy S1 nuclease from ClonTech (Takara) but it should work fine regardless of what supplier you buy it from. Takara also sells Mung bean nucleas. I would recommend trying Mung bean nuclease first as its application in blunt ending dsDNA, dsRNA and DNA/RNA hybrids is very well established.
Use Exonuclease T from NEB:
https://international.neb.com/products/m0265-exonuclease-t#Product%20Information
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