impossible gene ! - (May/16/2007 )
I'm having big trouble with the analysis of a gene using power master mix of Applied Biosystems with SYBR technology.
So far I tried 9 different primer-pairs for analyzing my gene. For each primer pair I first performed an RT-PCR to test if it works well. In 7 cases I found a good signal and I used primers in qRT-PCR. In 5 cases primers formed dimers and even changing melting temperature, primer concentrations (300-300 300-50 50-300 50-50) and using DMSO it wasn't possible to avoid dimer formation. In two cases the reaction seemd working well. I choosed the one with an higher signal even if it had an higher background compared to the other pair. I performed the standard curve and everything worked as expected. When I analyzed my samples some of them behaved well (single specific peak) other not that much since a dimer peak appeared.
At that point I chosed the second pair of primers that worked well and when I amplified dimers appeared as well. So I try a DMSO gradient and I observed that at 2 and 4% DMSO a single specific peak was present.
Again when I performed the standard curve (using the same sample) I found a nice dimer peak .
Also in this case I tried to change the melting temperatures, but it seems that sometime it works sometimes not.
Now I'm checking on an acrylamide gel if the problem could be degraded .
Has any of you a possible explanation to such variability in my experiment.
I don't know if it could be related to the gene since for other genes I've analyzed in the last month (using the same cDNA) no such problem emerged.
I checked if the primers blast in a conserved region and in one case it is so, but this eventually could influence the presence of aspecific products but not dimers.
Have you checked all your primer pairs to make sure they are not forming primer dimers? Since SYBR binds to any dsDNA then if they dimerise at alll then this can easily override the signal of the proper product. Since you've used the cDNA with other primers and these have worked fine then I'd suggest really looking at your primers again. Make sure they cross an exon boundary to avoid gDNA contamination and it's worth doing a BLAST search to see exactly what else they could possibly bind to. A colleague of mine spent 4 months on qPCR only to find out he was amplifying a pseudogene which showed transcript homology to his gene of interest.
I used to screen genes for mutations and have made MANY primer pairs that work well. The few times, where it did not work
was due to a GC% of 85 - 90 %.
Try to use mfold on the internet to see the secondary stucture.
Thanks guys I will pay attention to everything you told me