Hi All,
I isolated RNA from human cell line using TRIzol reagent and sent isolated RNA for RNA-seq analysis. The company sent me a QC report showing that some of my samples has additional peak at 28s region. (See attached). I am just wondering what is the cause of this peak and will that influence my RNA-seq analysis?
Thank you all :)
I would guess that this is fragmented genomic DNA, which can sometimes end up in the aqueous phase during a Trizol prep and will then co-precipitate with your RNA. This is most likely if you are using Trizol to extract RNA from a buffered aqueous solution (which can increase the pH of the phenol mix a bit and promote DNA coming into the aqueous phase) or if you didn't use quite enough Trizol for the amount of sample. I will say it is unusual to see DNA contamination showing up as a sharp peak like this on the Bioanalyzer, it is more normal to see a long 'tail' of high molecular weight material and/or some interference with the 18S and 28S peaks, so I am not certain this is DNA, but I can't think of anything else it could be.
If these were my samples I would take a subsample (maybe pool small amounts of the samples which contain this peak) and DNase treat it to see if the peak goes away.
I agree with Laura Leighton - you can try treating it with DNase, or if it's some kind of adaptive-dimer situation, you can always try to perform standard AMPure purification to see if it will remove your problem.
Hello Yun Zhang,
I agree with Laura Leighton.
In addition, this kind of representation (Agilent Bioanalyzer?) is very classical. Your RIN at 9.10 is high (which is very good); however, the additional peak after the 28s region seems 'suspicious'... Did you use DNAse for the preparation of your RNA samples?
Hoping that this comment will be useful. Feel free to discuss or share some informations about your RNA-Seq studies.
Ludovic Dumont, Ph.D.
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