Hi Christopher,

As an alternative to denaturing gels, you can use DENATURING LOADING BUFFER (2X): add the corresponding volume to your RNA sample, heat at 65°C for 3 min and then keep on ice till loading.

 5mM EDTA

0.1% (w/v) bromophenol blue

0.1% (w/v) xylene cyanol

95% formamide

As a further alternative, if you heat the RNA at 65C 3 min and then put on ice till you load the sample, you get a very good degree of denaturation for routine gels. The key is to prepare the gels fresh and use fresh running buffer: this way you get the best resolution and you minimise the chance of RNA degradation.

Ethidium bromide binds to the nucleic acid grooves (however, RNA is single-stranded), so the better the denaturation the less secondary RNA structures, and the poorer the staining. But anyway, 4ug of RNA is a huge amount, so it could be that your ethidium bromide is degraded? How does it stain DNA gels or non-denaturing ones?

Unfortunately I haven't used denaturing gels (the buffer above fits my purposes well). If you used the buffer, I can say that EthBr and SafeView (similar to SYBRSafe) stain OK, and for increased sensitivity at very low concentrations you could try SYBRGold.

Cheers,

Ruben

Just for your information and maybe others having the same issue:

Midori Green Advance can be used to detect RNA in denaturing formaldehyde gels (however, with less sensitivity (maybe 3- to 4-fold) than ethidiumbromide).

Update: For high intercalation and good staining results, we used to add EtBr directly to the samples loaded onto the denaturing gels. This, however, resulted in high background when combined with short running times (EtBr is stil present at the top of the gel). In comparison to this, we got much better results when the gel was stained in an EtBr bath after the run (0.5 µg/mL) and washed one or two times with H2O afterwards (15 min per step).