Are you sure that you have only one RNA species? if so, you might have some complex secondary structure. Try running a urea gel instead or heating to 55 ºC your RNA.

This in vitro transcript was first run and purified from denaturing urea-PAGE gel. Yes, I do believe I have more then one RNA conformer. However, that does not explain the way my RNA bands migrate through non-denaturing gel: the smudging and smearing...

In a non denaturing gel RNA may run aberrantly and at 1000 nt it is likely to have a potentially complex secondary structure that cannot be resolved without heat and the presence of a denaturant. My advice would be to run a denaturing UREA gel and dissolve sample in an appropriate RNA loading buffer, heating the sample prior to loading.

Looking at your gel, it seems that something is retarding RNA migration, suggesting several folding options for your conformers. I would try a 4-20% gel, but I usually work with very small transcripts...

Try loading less RNA and running the gel slower. Non-denaturing gels should not be run hot.

The transcript is long and it will fold to form secondary structures that are not "forced" to open in a non-denaturing gel. Why do you need to run the transcript in a non-denaturing gel? Is this kind of migration pattern problematic? If you need to confirm that this band contains the transcript in which you are interested one option is to cut the band and "load" them on a second denaturing gel in which the transcript will denature and migrate according to its size (and not according to the structure). Attached I send you a publication that give additional information about this technique, please look at Figure 6, may be it can help you.

Article A kissing-loop interaction in hammerhead viroid RNA critical...

Native gels with RNA can be tricky. I assume you are using a native gel because you are looking for interactions that you could not see on a denaturing gel. Bands can be smeary or streaky on native gels because the RNA structure you are observing is not stable during running of the gel (for instance, not stable under force of electrical current, not stable in ionic conditions of gel, changes structure on a timescale faster than the gel you're running). Sometimes it works right away, sometimes it takes a lot of troubleshooting, and sometimes it never looks quite the way you would like.

1. Make sure it doesn't get too hot. There are little liquid crystal thermometers you can even stick to your plates. Some people have also told me it is not good if it is too cold - i.e. recirculating chilled buffer with your apparatus at R/T may be better than putting the whole thing at 4C.

2. The buffer. What buffer system are you using? Sometimes it helps to use a native gel buffer with Magnesium in both the gel and the buffer.

-0.5X TBE (run at room temp or chilled; I have used this for super stable interactions - like separating duplex and ss substrates)

-Tris Acetate (a classic, usually the buffer or the entire gel is chilled. Add some Mg to help stabilize your RNA. Unfortunately, you must recirculate buffer regularly (Mg precipitates out))

-Tris-HEPES (A nice mixed buffer system, usually the buffer or the entire gel is chilled. Add some Mg to help stabilize your RNA - recirculating only necessary if that is how you chill your buffer. My personal favorite for large structured RNAs)

-- You can try 1-10 mM Mg. One of my colleagues in the past actually got the best gel with slightly different concentrations in the buffer and the gel.

3. The power. You may need to run this gel really slowly. If you look up nice native gel protocols, they usually suggest x V/cm. The amount of electricity you use is important not just for how hot it makes the gel, but also for its potential to destabilize RNA interactions (it is a force acting on the RNA). At the same time, don't run slower than suggested as you don't want to run into timescale problems.

4. Making nice looking bands. Make sure your wells are REALLY REALLY clean and REALLY REALLY beautiful. Have something to make heat even throughout the gel (metal plate, etc). If you have an empty lane next to your sample lane, you can put a little loading dye in it. Note that sometimes changing the volume of the sample or the size of the well can change the way your native gel looks.

5. Sometimes you just get gremlins. Decide how much time you want to take to try to get a beautiful gel, and what is good enough for your purposes. Don't get stuck rerunning gels forever!

Good luck!

Thank you very much for your suggestions! I will try to resolve that issue by monitoring the quality of my gel and the conditions I use to run it.

One more thing that was not directly addressed... too much salt in the sample can cause what i call "devil horns," the which is the outsides of the band running slower, and also streaking in the middle. So do as was suggested and try some different buffer systems and only have as much salt in the sample as is necessary.

Thank you very much Keith! That could be it. I fold my RNA in folding buffer (10 mM NaCl, 5 mM MgCl2, EDTA) before I run it. I noticed that folded samples always have "devil horns", while unfolded don't, so I bet my folding buffer is the reason. I will check that first, and then I will check other options suggested by Amanda.

Hi,

you can also try to migrate your RNA in 0,8 % of agarose in the TB 0,5x and Mg2 + 0,1mM in 4°C. Make your run with 8V/cm of gel. This is the best gel to see long RNA with several configurations. good luck.