RNA integrity - strange bands on the gels

[Luisinlab]

I run some samples from plant tissues side-by-side on a non-denaturing 2% agarose gel (in TBE) and I have some samples with higher-weight ribossomal bands than others. The intensity ratio is OK in every sample and there are no smears. What is happening? (The samples are from similar tissues from the same species!!)

Help me please. I need to know if some of this RNA is not suitable for RT-PCR!!!

see attached picture (line 1 -ladder; lane 2 (suspected sample)
What is the difference between line 3 and lines 4-8???

[admin]

RNA GEL

Hello there and welcome to the forums!
My guesses in no particular order:
1) sample contaminations
2) salt differences
3) loading amount differences

if you are sure you didn't contaminate your samples then did you extract them all the same way at the same time?

please tell us what you found
cheers!

[Luisinlab]

They were not extracted at the same time but the solutions were the same...
Salt differences? What do you mean by that? (different concentrations; for sure! but that explains the lower weight of the bands?)

[neja]

Hi

How did you store the samples between isolations, how were you preparing the tissue for isolation, was it unfrozen for a longer period of time ...? RNA is not as stable as some of us would like it to be when we are working with it, so it could be that you didn't handle the samples correctly and it was partialy degraded. If you are 100% sure that you can exclude contamination (you might have had something in your buffers from 1st extraction on) this might explain what is going on.

neja

[admin]

Originally Posted by Luisinlab

They were not extracted at the same time but the solutions were the same...
Salt differences? What do you mean by that? (different concentrations; for sure! but that explains the lower weight of the bands?)

Yes salt differences can make large differences in the migration of RNA or DNA in agarose gels. This may have been your problem.

Did you purify samples or desalt them prior to running them?

[mgks]

I've ran my samples from fish and I've got three bands, two more or less normal and a third one strange on the top. Do you know what can it be?