I am working on RNA-protein adducts, and trying to get rid of the unprotected RNA regions (that is not occupied by any protein). I am using RNAse A/T1 to degrade the RNA. For how long should I treat my samples with RNase to obtain efficient degrdation, without degrading the protein protected region of RNA. Data sheet recommends 10-15 min.
Greetings,
I've used RNase A to degrade dsRNA encapsulated on a VLP, depending on the stability of your RNA-protein complex, RNase A shouldn't interact or degrade the protected RNA, specially if it's on an hydrophobic pocket, wich seems very likely to be your case. Try making aliquotes and test for 10, 12.5 and 15, that should give a glimpse about how much is too much. Be aware of the composition of the buffer, as chaotropics would make your complex unstable, bringing the protected RNA in contact with the surrounding buffer, yielding in probable digestion by RNase A.
Note.
Depending on the nature of the RNA-protein mix, you should take in count that RNase A does digest ssRNA and dsRNA when low NaCl concentration is used, but becomes ssRNA specific when NaCl concentration is >150mM
Good luck
Hi, Mansi Srivastava
Although it is not my field of expertise, my best shot would be to tell you to make a small pilot test, If you have enough sample to do it.
I would do a 5 tubes with the same amount of RNA:protein complex as you have and add the standard amount of RNAse recommended by the company protocol. After that, i'd take one sample at each 5 min (or 5 min, 10 min, 20 min, 45 min and 1 hour) and then do an RNA gel electrophoresis and check the RNA integrity. If it works, choose the best time (where it gives you the best RNA decrease/elimination). Just an idea.
A little bit like in this CSHL protocol available at the website: http://cshprotocols.cshlp.org/content/2014/6/pdb.prot080788.full
As the other scientists here told you. If your protected areas are inside a pocket with access restrictions, RNAses may be not able to degrade it. But, anyway, you will need to check it. I guess.
Hope it helps.
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