Hi everybody,
I carried out a qPCR in order to quantify expression level of two genes under different conditions. I used three pairs of primers, one for the endogenous control and two for the GOI, I did not design but they have already been used by a colleague before.
I added NTC samples and -RT (RNA after DNase treatment).
I got amplification in both, NTC and -RT samples. The peaks of these samples showed by the melt curve matches exactly the peaks of the unknown samples and even Ct value is not that high but almost the same of a few unknown samples.
I would suppose a primer dimer formation because of the amplification in NTC rather than DNA contamination but I'm just at the beginning with qPCR and I would love to hear from anyone else using it opinion, advice and suggestion.
Hello,
I may have a few thoughts connected with your question.
1. Have you asked your collegue about his results? Did he encountered the same problem with amplification in NTC?
2. Does the mentioned problem refer to all 3 analyzed genes or only for one of them?
3. If the product is visible in NTC, RT- and unknown samples, there is high possibility, that these are primer-dimers. If it would be DNA contamination, there should not be a product in RT- (of course, assuming that DNAse treatment was complete).
It might be a few things. Firstly, it can be contaminated primers or DNA in your sample. After DNase treatment did you run the RNA on a gel to confirm that there is no DNA? But this should not be a problem in your NTC, so I would assume that it is contamination in primers. Go back to your stock primers and prepare new working solutions and see if it changes.
Marcin, of course I ask and I fount that he has never add neither NTC control nor RT- so I can't compare our results. Amplification is showed in negative controls for all the three primers pair and as you said, I'm also thinking it's a primer dimer formation but how could I solve the problem?
Henry, I don't run RNA samples after DNase in order to maintain a backup for a second assay if anything goes wrong. Would you strongly recommend that step?
Thank you very much for your answers.
I always run a gel after RNA extraction and DNase treatment just to make sure, alternatively you can do a control PCR using genomic DNA primers to confirm that there is no amplification before doing cDNA. It is not crucial as most of the time the DNAse treatment will be effective, but if you have problems later on it can be difficult to figure out where the problem is.
If you keep on seeing amplification in you your NTC then I would suggest to prepare all your solutions and primers fresh that you use for the qPCR, run a new NTC and if it is still a problem it is most probably something with your primers. This you should puck up when you do your melting curves though? Also it should be reflected in your Ct values.
If all 3 primer pairs amplify in NTC and the product is the same in your NTC and analyzed samples...then maybe you should redesign your primers? If above-mentioned ideas won't work (I mean, trying to prepare fresh solutions of primers for example), then such experiments cannot be analyzed properly. If this is something non-specific, then maybe some optimization might help, but I doubt it honestly in this particular case.
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