Difference between TBE- and Denaturing Agarose Gel Electrophoresis of RNA - Gel for RNA (Sep/15/2008 )

Hello everybody,

since some times I am working with RNA and after the isolation I make a gel to see if my RNA is good and clean. All the time I used TBE-agarose-gel, but today my doctor said to me, that I should use a gel only for RNA. So I make a gel with MOPS-Puffer, agarose and formaldehyd.

Now I have a question: where is the difference between the two gels?

Thank you for your answer.

Greetings
Mephi

TBE-agarose gel is a Native gel it helps you to know if there is any RNA or not. But to know the quality, the size, etc. a denaturing gel is need. RNA have loops and pin so the formaldehide/MOPS make it "straight" so is easier to distinguish between some of the RNA types. For a good recipe of denaturing gel go to ambion website. The MOPS buffer is sell at 10X just need to dissolved 1X with DEPC treat water (MOPS buffer is a little bit hassle to prepare).

It really depends on what you want out of the gel. Merlav explains the difference well, although I disagree about "quality" only being assessable on a denaturing gel.

If you are checking that your RNA is intact for reverse transcription or something similar then a native TBE (or TAE) gel is sufficient. From the statement "RNA is good and clean" I would guess that a TBE gel + spectrophotometry, which gives you a measure of template concentration and whether there is any contamination (protein or solvent), meets your needs perfectly.

Denaturing gels are a lot of extra work, extra risk (to you) and are difficult to get high quality images from. If you are wanting to do a northern then use a denaturing gel. Please make sure that everything with formamide and DEPC is done in a fumehood. If you don't use deionized formamide your RNA will be degraded. I second Merlavs suggestion of using pre-made MOP's (I use the sigma mix) if you need to run a formamide gel.