I have to run some RNA samples from 0.5X TBE (tris-boric acid-edta) native gel (non-reducing condition). I have consistently experienced smearing associated with RNA bands. Also the smear appears to be occupying the entire lane. this makes me think the smear may be due to some systemic factors (instead of degradation), but I don't know what are the causative factors. any idea how to improve the gel looking and to get rid of the smear?
enclosed a typical example:
lane 1&2 are viral RNAs extracted with trizol/chloroform/IAA;
lane 3 is viral RNAs extracted with trizol/column wash
besides the predominant two viral RNA bands, there are heavy smears the entire lane 1 and 2, starting from gel well. in lane 3, since it's column purified, the smear seemed to be better, but still plenty in the low mw range.
last three lanes are EGFP rnas, in vitro transcribed with T7 and purified with column. once again, there are smears below and after the RNA bands (between RNA band and the undigested DNA band).
Hi Tommy
There are RNAs and also sufficient quantities for downstream assays, I recommend you for better figure run your RNA samples on a 2% Agarose gel but each and everything for this should be RNase-free (From gel tank, buffer, loading dye, comb, etc.) .Always try the manual based Trizol method for high-quality RNA.
Have you tried purifying you RNA after isolation by ethanol precipitation?
Like you observed, most of the smearing comes from samples that you did the trizol extraction with, but not so much in samples that went through a column.
With trizol extraction, there is likely some residual DNA contamination.
Also, try using a higher % gel. 0.5% is good for DNA, but too low for RNA.
I agree with both above answers and also i would like to add that , you should be fast and precise enough to isolate RNA. Use DEPC treated tips, appendroffs, and while making 70% or 80% ethanol for precipitation make it in DEPC treated water. Wear gloves. make 2% gel.
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