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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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PhillipComment
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Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.07-01-2026, 11:43 AM
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I’m not a sequencing expert. I’m a purification scientist who uses NGS to evaluate workflows my group develops. With this perspective, we think about the sample first and the NGS workflow second. The sequencer is an exceptionally honest reporter, but it can only report on what you give it, so whether you get clean, interpretable data from an NGS workflow is largely determined before you begin.
Here are nine questions we think about, in roughly the order they matter, before...
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