DNA contamination of RNA samples for qPCR...weird results! - (Nov/30/2010 )
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!
Im unsure of the first part of your question, but as for the second DNA contamination issue.
I had DNA contamination issues when using RT-PCR also, used the trizol method as well as the DNAse treatment(ROCHE) but still showed bands in my RNA.
Resolved this problem by using DNA free from Ambion, i think its also called Turbo DNAse free. Its a modified DNAse enzyme that is apparently 480fold more effective than other (well thats what the box says anyway) and uses a sedimentation puty/spin down step to remove the enzyme after treatment (as opposed to heating to deactivate which may damage the RNA)
It worked completely for after 1hr treatment, not a band to be seen, but it is a bit pricey.
Good Luck
You can also try manganese buffer instead of the usual DNAse buffer, works better for qPCR it seems. Just stumbled upon it... http://www.biotechniques.com/multimedia/archive/00011/97226st05_11175a.pdf
rsm
Chris22 on Wed Dec 1 14:43:18 2010 said:
Im unsure of the first part of your question, but as for the second DNA contamination issue.
I had DNA contamination issues when using RT-PCR also, used the trizol method as well as the DNAse treatment(ROCHE) but still showed bands in my RNA.
Resolved this problem by using DNA free from Ambion, i think its also called Turbo DNAse free. Its a modified DNAse enzyme that is apparently 480fold more effective than other (well thats what the box says anyway) and uses a sedimentation puty/spin down step to remove the enzyme after treatment (as opposed to heating to deactivate which may damage the RNA)
It worked completely for after 1hr treatment, not a band to be seen, but it is a bit pricey.
Good Luck
Thanks! I think I will try the Turbo DNAse free. At this point, it doesn't really matter the cost since we need to finish this project. My other problem, well, it's that supposedly there's gDNA contamination but I'm not able to see the product from my no RT controls on a gel even though I get Ct values (between 15 and 25) for them
It kind of makes me doubt that there's contamination yet how else can I get a Ct value?Thanks again for you help!