I extracted RNA from soybean with a Zymo Research column extraction kit (with Tri-Reagent) with an in-column DNAse I digestion step. I got good nano-drop results with good 260/280 (2.03) and 260/230 (2.1) readings and a concentration of 876ng/ul. When I ran the RNA on an Agarose gel I got the two bands expected in an RNA gel but also I bright band above the well. This could be gDNA contamination but I would expect the this to be below the well (even if marginally ), not above it.
I ran the gel using an RNA electrophoresis specific loading dye that already contains EtBr in it. Is it possible that the excess EtBr is dissociating into ethidium (positive) ions and Bromide ions and the ethidium ions are migrating towards the negative pole above the wells, resulting in a fluorescent band above the wells?
Dear Cameron, first of all it seems that you had overloaded gel. I agree with@Utsa. Try to decrease sample volume at least in twice. About an artefact on the top of well, it is difficult for me to be sure, but I can suppose that it could be part of sample that could not enter in gel because extra volume. But I do not know exactly.
Ragarding the bright band in well, which kit did you use for purification? If column-based it could be genomic DNA, if Trizol or simillar, I do not know. And it is very strange for me - direction of top bands. It seems that they running to the top??? To +
An update: it does seem to have been an issue with the loading dye. I ran the same RNA sample using the RNA loading dye (with EtBR in it) and using standard loading dye i.e. with no EtBr in it (first picture) and only the well containing the former had a bright band above it. I do suspect it is the EtBr dissociating because I recall a while back that someone suggested adding EtBr (i.e. not as part of the RNA specific loading dye) directly to my RNA samples to better visualise my sample and got the same thing (see last picture). Attached as well are close ups of each of the lanes in the first picture to adjust for differences in exposure requirements of each lane (second and third pictures).
As as solution, I simply add the EtBr to the gel and not the sample (although perhaps adding to the sample at much lower quantities will also yield good results).
If you add the EtBr directly to your samples you will get the retro-projection of the EtBr. I discovered this once I did 2 rows of wells in the same gel and run it (just as a proof). I essentially hide the upper row with the EtBr retro-projection of the bottom row. I personally prefer the EtBr directly on the sample thus the background noise is erased. You should not mind about that upper noise, although try not to overload as this shadow could also cover the lower part of the well and you should keep this clean in order to check gDNA contamination and other issues.