doing a polyA capture twice - does it work?

Go to the profile of molbio1234
Jan 24, 2018
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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?

3 Comments

Go to the profile of relaxin
relaxin 6 months ago

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.

Go to the profile of molbio1234
molbio1234 6 months ago

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?

Go to the profile of relaxin
relaxin 6 months ago

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.