I recently extracted RNA from a mixed population of white blood cells taken from a selection of adult and juvenile grey seals (Halichoerus grypus). I have initially extracted RNA from 13 samples. The RNA was extracted using TRI Reagent and quantity was determined using a Nanodrop; all samples had between 500 – 1500 ng/µL, 260/280 ratios were between 1.90 and 2.1 and 260/230 ratios were between 2.0 and 2.50.
However when I ran the RNA on an agarose gel there appeared to be three bands (please see image). All 13 samples show the third band when ran out on an agarose gel.
I ran the RNA on an Agilent Bioanalyzer chip and still the third band was visible with a clear peak next to the 28S peak on the electropherogram (please see the attached file).
I am considering running a denaturing gel to see if this removes the third band. I thought that the third band could be a secondary structure of some kind (which I thought might be removed by heat denaturing the RNA during the Bioanalyzer prep.).
I intend to use the RNA downstream for qRT-PCR. My worry is that the third band represents a structure that will interfere with the efficiency of enzymes and primers in my reverse transcription reaction and qRT-PCR.
Has anyone had a similar experience with mammalian white blood cell RNA or are there any suggestions that explain the presence of the third band?
Many thanks.
Hi, that is exactly what you expect. These are the bands from ribosomal RNAs28S, 18S, 5.8S and 5S which are always prominent in an (eukaryotic) RNA extraction. 5.8 and 5S RNA are smalles one and run at 250bp (check that) and are sometime missing depending on the columns of the kit you used. Thus, nothing to worry about. Otherwise, you will see in your qPCR if the efficiency is correct (because you do a standard curve). you will lose too much of your sample by checking the quality in so many ways.
Good luck!
Hi, that is exactly what you expect. These are the bands from ribosomal RNAs28S, 18S, 5.8S and 5S which are always prominent in an (eukaryotic) RNA extraction. 5.8 and 5S RNA are smalles one and run at 250bp (check that) and are sometime missing depending on the columns of the kit you used. Thus, nothing to worry about. Otherwise, you will see in your qPCR if the efficiency is correct (because you do a standard curve). you will lose too much of your sample by checking the quality in so many ways.
Good luck!
Hi Sinje, thank you for your response! The third band is much higher up than 250bp; it sits just below the 28S band. Is it possible that it is a 26S or 23S rRNA?
Hi, I never had prokaryotic contamination but my samples were always taken in very clean enviroment (hospital or special animal houses). But I would expect that the contamination wouden't be visible on a gel. Are you sure that the middle band is the additional one? 28s runs between 1.5 and 2 kb and 18s at 800bp (DNA marker), everything above is likely to be genomic DNA. Did you perform DNA digestion during RNA isolation?
Hi again, yes I treated my RNA with a genomic DNA elimination buffer (Qiagen) - the third band is still visible on the gel. I did consider that the third band was genomic DNA contamination but I was not sure if it could appear as a distinct band on an agarose gel (I would expect more of a smear above my 28S band). When I put my RNA on a Bioanalyzer chip the third band appeared below the 28S band in the majority of samples.
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