We have isolated RNA from skeletal elements dissected from a mouse pup. But when we run the RNA sample on 1% agarose gel we get three bands. RNA isolation was performed by QIAGEN RNeasy mini kit. We did not observe this kind of effect before with RNA isolation by using the same kit. Can any one explain the reason why we get three bands in our RNA isolation rather than normal two bands. Please look into the picture for reference.
I have seen others have this problem. There are a number of possibilities. (1) fragmented 28s. (2) contamination from the components used to make and run the gel (3) DNA-could be an incomplete DNase digestion or lack of. Keep back some of this sample to compare to after eliminating these variables individually. If that gives no better results, then I would want to use new everything, the water, the kit, the sample, etc. and start over.
P.S. The RNeasy micro plus kit may be better for some types of samples.
Which ladder did you use?
Are there any very highly abundant transcripts in bone, much like hemoglobin in blood?
This wasn't a denaturing gel I take it?
It looks to me like there are 4 bands in your gel. I've observed similar when extracted from mixed eukaryotic/prokaryotic sources.
Assuming you ran a TAE (non-denaturing, as Jordan correctly asked) gel and looking at the smear I would say you have quite some degradation, so as suggested by Ginger the 28S might have been split in two or more larger pieces before smearing. Next time instead of a DNA marker that runs quite differently from rRNA I would use a control RNA that you are sure is of good quality.
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