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I have run an in vitro transcription reaction and produced some RNA of a single species and definite size. I would like to visualize it to check if the reaction worked.

Can I do this on an agarose gel? Is it sufficient to simply make a 1% TAE-agarose gel with EtBR that you would use for routine DNA electrophoresis, and run this? Is it necessary to take special measures against possible RNAses in the gel or the running buffer?

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  • $\begingroup$ Include in your agarose gel 4% household bleach to inhibit rnases. It's a much easier alternative to PFA and just as effective. $\endgroup$ – rhill45 Oct 9 '14 at 7:30
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You have two possibilities: When you only need a quick check if your RNA is ok and you indeed only get one band, you can try a "quick and dirty" method. Heat the sample for 5 minutes at 65°C and then immediately cool it down in an icebath and keep it there until loading. By doing so, you melt up the secondary structure of the RNA and keep it in this state. You will most likely then see only one band.

If this doesn't work or if you want to use a RNA size marker, there is no way around denaturating gel electrophoresis. Here you denature your sample in the loading buffer and then run a denaturating gel in which prevents the secondary structures. There is a good paper about this:

The classical method using formaldehyde (which is more dangerous in handling) is described in the following paper and also in the "Molecular Cloning: A Laboratory Manual" book by Maniatis and colleagues:

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RNA is amenable to folding, which produces secondary structure and alters its mobility (compare to supercoiled DNA). Therefore, if you don't denature your RNA appropriately, even a single species will produce a smear.

A common strategy is to incorporate denaturing chemicals such as formaldehyde into the procedure. See this Life Technologies protocol.

Apparently, you can also just make a standard DNA gel with some added bleach. I haven't used this technique, but they have some samle results.

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    $\begingroup$ I use a 1% nondenaturing agarose gel with ethidium bromide. I use Better Faster Media LB buffer so I can run a high voltage and get my gels done faster. I usually see 2 bands on the gel due to RNA secondary structure, but that can be fixed by heating it to 65C for 5 minutes, followed by chilling on ice for 5 minutes. My RNA may be different from yours, so you might not be able to get a single band with this trick. What I haven't been able to do is run an RNA ladder on nondenaturing gel, but I haven't really tried that hard. $\endgroup$ – user137 Oct 8 '14 at 0:46

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