doing a polyA capture twice - does it work?

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doing a polyA capture twice - does it work?

Postby molbio1234 » Jan 24 2018 4:53 am

Has anyone ever done a poly-A capture twice on mRNA material? Has your recovery been good? I have mRNA enriched material from another source and I need to use it as input into another protocol but the input requires it to be in the protocol specified buffer solution. I would like to recapture the material so that it is eluted in the protocol specified buffer solution (which contains enzymes and some cocktail of reagents, not just a simple buffer). In essence, I will be doing the polyA twice. Has anyone done polyA capture twice and has it worked for you?
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Re: doing a polyA capture twice - does it work?

Postby relaxin » Jan 26 2018 4:44 pm

I do not think eluting mRNA with a buffer containing other stuff is wise, as it may interfere with elution. If you want change of buffer, you may ethanol precipitate with non-RNA carrier, rinse, dry and re-dissolve in new buffer.
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Re: doing a polyA capture twice - does it work?

Postby molbio1234 » Jan 28 2018 12:51 am

Hi relaxin, the mRNA I received from the other source has been eluted in water. I am not looking to do a buffer exchange. Because I have to input the mRNA into another protocol, I need to repeat polyA capture. Do you have experience doing polyA capture twice and how well does it work?
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Re: doing a polyA capture twice - does it work?

Postby relaxin » Jan 30 2018 7:05 pm

The recovery is not 100%. So you will lose mRNA if you do a second polyA capture. There are small spin columns to minimize loss. You still need to elute with water.

If there is not problem in RNA concentration, you can just add concentrated buffer (2X or 10X) to the RNA for the downstream application.
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