The protein X I am studying may related to RNA binding, so I am interested to test the RNA that can be bind by protein X. I read some paper, and found that there are several kind of methods and protocols.
1.I am not sure to choose RNA-IP or RNA-CLIP, which is better for my experiments? And can you recommend a good protocol?
2.what is the essential difference between RNA-IP and RNA-CLIP?
3. Actually we supposed that X may related to splicing? But I have not done RNA-seq, and have not found the RNA targets. Therefore, I had another problem, even I successfully do RNA-IP, I do not know which targets to test? Any suggestion? Need I do RNA-seq to find out the RNA targets first?
Thanks.
According to some helpers' answers, CLIP will be better. And I checked some protocol, in Abcam CLIP protocol, it mentioned "irradiate once with 150 mJ/cm2 at 254 nm using a stratalinker", I am wondering what is the 254nm mean? And it did not mention how long for crosslink.
For the UV crosslink step, somebody who done before can help to suggest detailed condition, including energy and time. many thanks.
the best should be by CLIP approach...after UV crosslink, RNA Binding proteins and RNA are covalently linked (in this case, you can increase stringency by increasing concentration of detergents and/or salt in your wash buffer and increase specificity of your IP)....you can read papers from Darnell R lab about protocols.
Dear Sue Yu, I would suggest you to do CLIP, because with this method you can capture not only the strong interaction but also the very weak ones, and you reduce the background because you can use very stringent conditions in the washing steps. Independent of the method you choose, it is very important that you have a good antibody against your protein. If you don't have one, then I would suggest you to create a stable cell line with an inducible tagged version of you protein (like that you can control the level of expression), and use and antibody against the tag (FLAG or GFP as an example). To be sure that the peaks you find (potential bound RNAs) are real, is very important that you set a proper background control. As an example if you work with a stable cell line with a GFP tagged protein, then you can do the CLIP as well with a only GFP cell line, or an unspecific antibody.
CLIP followed by RNA sequencing have been successfully applied to identify RNAs as well as the exact RNA motifs recognized by RBPs in cultured cells (König et al. 2011a;Ascano et al. 2012).
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