I sometimes use denaturing gel electrophoresis on a preparative scale to purify RNA produced by in vitro transcription. The major issue with this is that the sample after extraction from the gel still contains significant amounts of acrylamide, or acrylamide oligomers/polymers.

Exchanging the buffer using Vivaspins doesn't reliably remove it, precipitating the RNA also only reduces the amount, but can't remove the acrylamide entirely.

In some cases the acrylamide doesn't interfere much with the later experiments, but there are some experiments that won't work with acrylamide in the sample.

What are the options to purify the RNA after gel electrophoresis that would reliable eliminate acrylamide from the sample? Preferably they should be simple and fast, not something like a full FPLC/HPLC run.

  • $\begingroup$ May I ask why you need to purify your in vitro transcribed RNA? I've been making mRNA by IVT and have gotten good results without any purification beyond phenol:chloroform extraction and isopropanol precipitation. $\endgroup$ – user137 Dec 17 '14 at 15:13
  • $\begingroup$ @user137 I use the RNA itself for NMR experiments, I need it in a different buffer and without the DNA and protein to get good quality spectra. And anything else that is still in there will likely overlap some of the signals of the RNA. $\endgroup$ – user11595 Dec 18 '14 at 6:39
  • $\begingroup$ How much RNA do you need for NMR? I've always heard that NMR needs a lot of material for good signal. $\endgroup$ – user137 Dec 18 '14 at 16:05
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    $\begingroup$ @user137 Depending on what exactly you want to do you need about 0.2-1.0 mM protein or RNA in your sample in a volume of 300-500 microliters. And for many experiments you also need it 13C and/or 15N-labeled. $\endgroup$ – Mad Scientist Dec 18 '14 at 16:55
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    $\begingroup$ @user137 The RNA you would investigate with NMR is usually around 15-150 nt long, you don't see much with NMR for anything larger. And we simply do the in vitro transcriptions on a larger scale, typically around 10-30 ml. RNA is also almost always measured in Shigemi tubes, so the volume is 300 ul, and while 1 mM concentration is ideal, but you can't always get that much. $\endgroup$ – Mad Scientist Dec 18 '14 at 17:22

If you don't want acrylamide in your preparation, don't use it, as you will always have some carry-over in the solution. And you will not get rid of it completely, as it at least partly co-precipitated with the nucleic acids (it's used as a co-precipitation agent for this purpose).

I think the best solution is to use chromatography, either size-exclusion or charged resins. See these two papers for more information:

  • $\begingroup$ We also use chromatographic methods, but gel purification has certain advantages like better resolution. I don't need to get rid of acrylamide completely, reducing it significantly below the RNA concentration would be sufficient. $\endgroup$ – user11595 Dec 17 '14 at 10:04
  • $\begingroup$ Gel is easier and faster, chromatography has a better resolution and takes longer. Now you have to choose what is more important... $\endgroup$ – Chris Dec 17 '14 at 10:08
  • $\begingroup$ I suspect if there would be any residual unpolymerized acrylamide after the run. Heating should decompose acrylamide to gaseous products (ammonia, hydrogen and carbon monoxide). Seehere. I am not sure about thermal decomposition though (no solid reference). $\endgroup$ – WYSIWYG Dec 17 '14 at 10:30

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