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I want to fold precursor-miRNA into its secondary structure and then confirm it on gel. At first, I heat it in annealing buffer at 95°C for 5 minutes and cool down slowly. Then I run it on gel but the samples are at same position as the original dilution.

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  • $\begingroup$ What kind of gel are you using? pre-miRNA will mostly from hairpin loops (I guess), which don't change the overall size of the RNA by much (it's still linear, now just a bit 'shorter' but therefore 'wider'). I'd guess you need a relatively high percentage gel (native and probably better poly-acrylamide), with custom markers for your size-range) $\endgroup$ – Nicolai Jan 22 at 12:28
  • $\begingroup$ Your question looks incomplete. For e.g. you haven't mentioned how many minutes at 95°C. Please edit your question to all necessary details. I advise that you provide the full protocol that you are using. $\endgroup$ – WYSIWYG Jan 22 at 15:45
  • $\begingroup$ My gel is 18 % polyacrylamide ( 40% acrylamide; 1M Tris, 1M Hepes, 1M EDTA, 1M MgCl2, TEMED and APS) in 1X Hepes (containing 1M Tris, 1 mMEDTA, 30mM MgCl2) $\endgroup$ – Javaria Feb 16 at 9:08
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Disclaimer: Although I have attempted northern blots for miRNA, I haven't actually done the kind of experiment you are talking about.

First of all you need controls.

  1. A negative control: a same length RNA that will not fold (you can introduce some mutations in your pre-miRNA sequence to disrupt the base pairing). You can use the miRNA secondary structure prediction tools for rationally designing such controls.
  2. A positive control: perhaps a folded pre-miRNA that is crosslinked. Frankly, I haven't come across anyone using such crosslinking agents to study RNA secondary structure. I had however, seen some old papers on this topic (Wagner and Garrett, 1978, Rabin and Crothers, 1979). You may also try some conditions (salt concentrations etc) under which the RNA will fold (studied using biophysical assays).

Now, you cannot run agarose gels. You would need long polyacrylamide gels ~15%. You also need to use the right buffers. 0.5-1x TBE supposedly works. You may want to run your sample in two conditions: denaturing (with 8M urea) or non-denaturing. You can refer to this protocol (Petrov et al. 2013).

Your positive control should migrate faster than the negative control. If your RNA is folded (fully or partially) it should migrate between the positive and negative controls in the non-denaturing gel. In the denaturing gel, your RNA should migrate at the same level as the negative control. Positive control (if it works) should not be affected by urea.

Also, the gel heats up during electrophoresis which in turn can dentature RNA. You may want to run the non-denaturing gel in the cold room.

Having said all this, I would advise that instead of putting in so much effort in gel based assay you can use more sensitive equipment to study RNA folding. There are many biophysical studies on in vitro RNA folding. You can check them out.

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