I am extracting RNA from frozen human PBMCs using the Trizol Reagent method.
Hi, did you see only a bigger band near the wells? Did you see only a band and what size? Without an image it is difficult but from your description I didn't understand if you had only a DNA contamination or your extraction didin't go very well and you see only an RNA band
Are you denaturing your RNA prior to loading on a gel (or are you using a denaturing gel)?
RNA is tightly structured (ribosomes in particular are massive globular structures), so does not electrophorese as you might expect, unless you denature it.
This typically works well for me:
Mix RNA (1-2ul) with 6x DNA loading buffer and add formamide to make up the remaining volume: aim for at least 60-75% formamide in the final mix.
Heat this to 65 degrees for 5 mins.
Crash cool by transferring directly to ice.
Load on a standard agarose gel as you would DNA.
The heating helps unfold the RNA, and the formamide and crash-cooling prevent 2ndary structure reforming. It then stays denatured long enough to resolve nicely on a standard gel without any need for in-gel denaturants.
Hi Carole, amount of RNA loading into wells also plays a role here, as usually above or near 5 ug RNA loading will lead to the entrapment of your RNA inside wells. Also, if you are interested in the sizing info of RNA through using RNA marker ladders (28s, 18s, 5s etc.) then a non-denaturing gel won't be helpful at all, as the bands could be of milder intensities and their differentiation on size basis is not so easy then. So make sure you are running RNA onto a denatured gel as Sir John has mentioned for better flow and clarity in terms of the size bands. 60-65 V is enough running voltage for the RNA gel. It is important to know that if you just get a single band either of 28s or 18s, you can go ahead to use that RNA for downstream.
Regards...
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