As the RNAse H may not be 100% efficient at clevage, I want to seperate the cleaved from the uncleaved. I cannot seperate them on a gel due to their miniscule size difference of 15nt against a 1.9 kb product. As the cleaved against uncleaved would have different 5' sequences, is there a way to seperate them based on that feature. Also if one is capped and the other uncapped would it be more useful?
Thanks
There are cap-G antibodies you could use to enrich capped (uncleaved) RNA, but then you would also enrich the 15nt fragments on the 5 prime end so you would maybe have to size select these away first.... a bit cumbersome?
In general I would say that your approach is extremely difficult and there may be an easier way to get what you want by a different path. If I understand correctly, you have an RNA-DNA hybrid of ~1.9kb with a 15nt RNA leader on the second strand, and you want to separate the RNase-H-digested from the partially-digested and undigested species. There may be ways to obtain the information or analysis you need without physical separations such as electrophoresis, but that would be hard to tell from what you've told us. It would help if you could give a bit more information about your goals with the product, for example do you want to clone and/or sequence it? Please elaborate.
You can use biotinylated oligonucleotide probe complementary to this 15-nt target sequence. You may want to use 2'-OMe or LNA modification for the probe to increase Tm of the 15-bp duplex. After RNAse H treatment, add the probe then heat to 95 and slow cool down for the probe-target hybridization, then capture the duplex on streptavidin coated magnetic beads. The solution phase will contain only the processed RNA.
Thanks so much for the information. I have never done probe-target hybridization. Is there a way to elucidate this?
Hi Anjali,
Check out the links below:
http://guttmanlab.caltech.edu/protocols/RAP_Complete_Protocol.pdf
http://www.miltenyibiotec.com/~/media/Images/Products/Import/0002500/IM0002568.ashx
https://tools.lifetechnologies.com/downloads/dynalCat_Nucleic_Acid_Isolation.pdf
http://www.nvigen.com/docs/61002_MagVigen-Streptavidin%20DNA%20Capture_product%20sheet.pdf
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