I observe double peaks in RT-PCR for the primers that were validated using gel electrophoresis in other samples. I have used these primers on three different sample sets (about twenty-four samples in each) and the data was clean.
In this case, I am using sixteen samples and these primer dimers observed (ran it on a gel) were not consistently found in all of these sixteen samples. I tried for two other genes, these primers gave clean results for all the data sets including the other three data sets. Now I am really confused - because this clearly does not seem to be neither a problem of primers nor of the sample quality. Why are these primer-dimers observed selectively only in some samples? I am not sure whether to proceed with the data I got from the RT run.
Can anyone help me with this issue ?
It's possible that you have contamination in some of your samples that is leading to the double peaks. That would explain why you are only getting the double peaks in some of your samples and why you are not seeing primer dimers on your gels.
Are you sure the additional peak stems from primer-dimers?
Can it be that some of your samples have a sequence variation in the amplified region? This should be also visible in a high resolution PAGE (6-10%) as a double band.
Could you display a picture of your dissociation curve? Do you also have primer dimers in your controls (NTC, noRT)? Once, I had the same problem with some samples showing primer dimers and others not. I simply repeated cDNA synthesis (2-step qPCR) or RNA extraction and got rid of them. However, I don't have a clear explanation for that. Do you have the possibility to repeat one of these steps?
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