Intense background into wells when running RNA samples - (Apr/03/2008 )
I have been running RNA samples from plant tissues (leaves and stems) into denaturing (formaldehyde-based) gels. I quantified in advance the obtained RNA by spectrophotometry yielding amounts from 1.4 up to 2.5 ug total RNA/ul and a purity of 1.8 2.0 both for A260/A280 and A260/A230 ratios. Nevertheless, when visualizing the RNA samples (stained with EtBr), I am getting an unusual intense background into the well for all the RNA samples from leaves. I am not really sure whether such a background could be gDNA contamination since DNA usually appears as a single sharp band just below the well but not inside of it nor as a hevy intense background as showed in lines 3 to 7 of the attached picture. Lines 1 and 2 correspond to RNA from stems tissue and line 8 to RNA control. Both RNA from leaves and stems were isolated using the same stock solutions as well as the same protocol. Since no RNA bands are present in the aforementioned wells, do you think that background could be hindering the RNA to run into the gel??? By the way I loaded aroung 5 ug of total RNA per well.
Please let me know your comments, which could help me to overcome this constraint.
Thanks ahead of time for your suggestions.
PS: Total RNA was isolated using an acid phenol-based protocol.
Is that an agarose gel?! What's your fragment size. Most RNA work is done using denaturing TBE acrylamide gels. You may also have degradation. Are you using Rnase inhibitors during your extraction? What's your extraction protocol? Leaves are especially prone to RNA decay. Make sure that when you collect them you flash freeze them in liquid nitrogen and then grind the tissue to a fine dust with a mortar and pestel (put the mortar in dried ice or liquid N2).
Hallo, I was reading your message and watching the picture, but I think it is not probablle gDNA block migration of RNA, It is possible that there is no RNA in samples. You should prolong time of the migration and volatage should by increased. But it is possible gelelectrophoresis is not capable detect your samples just because it is under detection limit. Consider using another method as Real Time PCR
I run RNA in denaturing formaldehyde/agarose gels all the time. (Usually 1.5% agarose, in MOPS buffer). This is fine if your RNA of interest is relatively large. For smaller transcript analysis, acrylamide is better.
It looks from your picture like those center lanes contain nothing but DNA. (And maybe some degraded RNA.) You may try loading your gel again, but it looks like most of the nucleic acid concentration you detected in your samples was DNA.