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Hi I was looking to do an RNA fragmentation protocol using the Covaris versus chemical cleavage (i.e. Zinc ion & high temp). Does the Covaris shearing of single stranded RNA result in 5' OH and 3' PO4 ends? Or scrambled like the chemical cleavage protocol? (i.e. 2' PO4, 3' PO4 and cyclic 3' ends). Any ideas on the product formation? Thanks!
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I don't know, but I would think that you would have fragments that have 5'-PO4, 3'-PO4, both or neither. I think T4PNK should be able to fix this, though. (Though I have not tested this myself.)
Most enzymes that fragment RNA leave a 3'-PO4/5'-OH or maybe a cyclic 2'-3' PO4 linkage. So that might be a general paradigm. But sonication, being mechanical, might generate results similar to DNA. I went over a (rather old) manuscript that studied this:
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Phillip -
Hi Argonaute,
Most likely the ends do have a PO4/OH much like single stranded DNA shearing using Covaris AFA. The ends should be treated as described in the paper located at http://www.ncbi.nlm.nih.gov/pmc/arti...164-12-332.pdf
Thank you
HamidComment
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Thank you Philip and Hamid will read over the two articles thoroughly!Comment
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Hi everybody,,,
what is the purpose of RNA fragmentation????Comment
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RNA is fragmented either chemically, or physically for use in RNA-seq.
hamidComment
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thank you hamid,,,
I've read a book explained about two kinds of enzymes which break the links in an RNA chain (G-enzyme and U,C-enzyme). are they produced in human body????Comment
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I think RNAseA is usually isolated from cow pancreas -- so human pancreas would produce it as well, in all likelihood. RNAseA cleaves ssRNA after U or C.Originally posted by Laily View Post
Not sure if there is a human RNAse that cleaves after G. That is a characteristic of a fungal RNAse, RNAseT1.
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