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Help with isolating protein-bound RNA

Protein RNA Isolation

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#1 molbiolnew

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Posted 01 February 2018 - 12:03 PM

Hello.

We'd be very grateful if anyone could help us solve a conundrum.

We want to pull down all protein-bound RNAs in a cell's cytoplasm (mammalian cells) for downstream work in an unbiased manner (i.e. we don't want to limit ourselves to using antibody-based IP) because we don't know what proteins are interacting with what RNAs.

Can anyone think of a way of doing this without?

Many thanks in advance.

 



#2 bob1

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Posted 01 February 2018 - 02:25 PM

Pull down the RNA instead - cross link the RNA to the protein and proceed from there.

 

These are known as CLIP experiments and are not for the faint of heart (says someone about to embark on doing a variant...)



#3 molbiolnew

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Posted 02 February 2018 - 06:18 AM

Pull down the RNA instead - cross link the RNA to the protein and proceed from there.

 

These are known as CLIP experiments and are not for the faint of heart (says someone about to embark on doing a variant...)

 

 

Thank you very much. Do you happen to have a good CLIP protocol that works (how efficient is the X-linking) or should we just use a generic one....also, what is your preferred method for RNA pull-down after that (and how efficient)? We want to eliminate all RNA species that are not protein bound...

 

Thanks again.



#4 bob1

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Posted 02 February 2018 - 12:37 PM

I don't think the protocols are very efficient in general - it depends on which RNA pool you are looking at and abundance of the proteins you are interested in.  The cross-linking is reasonably efficient, but there are limitations in how much (RNA or protein) you can detect and how good your fragmentation protocols are. I would expect substantial losses at most stages of these protocols. I have heard of people refining these sorts of experiments to work on limited size clinical samples. RNA pull downs of polyA-mRNA are reasonably good these days, but will have carry-over of non-polyA mRNA. The best kits for this are not at all cheap and you can expect to use them rapidly as you need to start with large amounts of RNA.

 

Note that CLIP protocols are usually targeting detection of RNA species for binding of things like transcription factors to particular sequences. You will need to adapt for detecting proteins - talk to your mass spec people about this.

 

However, with the advent of mass spec techniques, it has become more feasible to do these sorts of experiments and get some meaningful data out.

 

I would go with a generic protocol and refine from there. In most downstream situations you will have specific questions that you want answered, and without the specifics you can't easily use a refined protocol for these. 



#5 molbiolnew

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Posted 02 February 2018 - 02:09 PM

I don't think the protocols are very efficient in general - it depends on which RNA pool you are looking at and abundance of the proteins you are interested in.  The cross-linking is reasonably efficient, but there are limitations in how much (RNA or protein) you can detect and how good your fragmentation protocols are. I would expect substantial losses at most stages of these protocols. I have heard of people refining these sorts of experiments to work on limited size clinical samples. RNA pull downs of polyA-mRNA are reasonably good these days, but will have carry-over of non-polyA mRNA. The best kits for this are not at all cheap and you can expect to use them rapidly as you need to start with large amounts of RNA.

 

Note that CLIP protocols are usually targeting detection of RNA species for binding of things like transcription factors to particular sequences. You will need to adapt for detecting proteins - talk to your mass spec people about this.

 

However, with the advent of mass spec techniques, it has become more feasible to do these sorts of experiments and get some meaningful data out.

 

I would go with a generic protocol and refine from there. In most downstream situations you will have specific questions that you want answered, and without the specifics you can't easily use a refined protocol for these. 

 

Many thanks. We'll take a deep breath and take the plunge.

Cheers.







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