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DNA contamination of RNA samples for qPCR


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#1 mls677

mls677

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Posted 24 November 2010 - 02:56 PM

Hi everyone,

I've been having this problem for quite a while and am hoping someone may be able to help me with it! I've transfected a cell line with a plasmid and then isolated RNA using a Trizol protocol from these cells, as well as cells that were not transfected, to look at gene expression. I performed qPCR and got Ct values from my no RT controls from both of the transfected cells and untransfected cells. These Ct values are sometimes lower, the same, or higher than the Ct values of my cDNA (just depends on the gene I'm looking at). I've looked at my NTC and they are clean, so I'm pretty sure that it's DNA contamination (even though my primers are all intron spanning and shouldn't be able to amplify from genomic DNA). The dissociation curves of my no RT samples and my cDNA samples show the same peaks (which I know to be correct for my products). I ran the samples on a gel and I see the correct band for my cDNA samples but there are no bands for my no RT samples! I'm assuming that it's DNA contamination, but I find it strange that I can't see it on a gel when I can see my cDNA samples (especially when the Ct values of my no RT are not high). I tried to clean up the RNA using Qiagen's RNeasy Plus mini prep, as well as treating with DNase from Invitrogen, but I'm still having this problem. Does anyone know what might be causing this? Is it really DNA contamination? Any help will be mostly appreciated...trying to finish up my masters but I'm at a standstill until this issue is resolved! Thanks in advance!




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