I do total RNA extraction from U87-MG cells and convert them to cDNA which I eventually use for gene expression studies but when I check the integrity of RNA by agarose gel it is showing 4 bands. I am sure that there is no DNA contamination as I did not observe any bands near the wells. Where am I going wrong? or is it quite normal during extraction using TRIZOL method?
I am observing bands something similar to the picture that I have attached.
There is no problem with that gel. Only show more RNA bands beside the ribossomal bands. By this picture I can tell you that the RNA has a good quality and you can continue with your experiments.
The RNA bands look distinct and intact, but I've never seen that extra band before and I extract with Trizol all the time. How are you denaturing the RNA before loading it on the gel? And you use a MOPS-formaldehyde agarose gel I presume?
Dear Sean,
I will usually denature RNA along with dye at 70 degrees for 10mins and I have been using urea-page gel but these days I am using agarose gel in depc water made 1x TAE
I've never tried running RNA in a regular agarose gel as it's not denaturing throughout. And I usually denature the RNA with a special RNA loading buffer containing standard glycerol-based loading dye, formamide and MOPS buffer (as I usually run MOPS-formaldehyde agarose gels). I think the band could be RNA with secondary structure that runs differently in the gel as it may not be completely denatured, or the RNA re-natured after your heating. Adding formamide during heating will help keep the RNA denatured until loaded and formaldehyde in the agarose gel will keep it denatured while running. Shall I send you my protocol?
You should show a size marker in order to allow judgement. I am convinced it is a denaturaytion/gel-effect as Sean Seitzer explains. Certainly, the picture does not tell clearly whether your RNA is ok.
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