two peaks in the melting curves, but only one product - (Apr/30/2008 )
Hello, I'm utterly confused with my qPCR dissociation curves and I wonder if any of you can help me. 
Basically what says in the title. My melting curves show 2 peaks, one at the expected Tm, another one around 10C lower. I thought the lower Tm would be primer dimer, so to check whether it was this or non-specific product I run my samples on a gel. Guess what, a beautiful single band of the expected size. 
Any suggestions as to what that second peak could be???? 
Thanks in advance.
Could you show us the picture of your melt curve and gel?
How long did you run the gel ... is the band under/just below your band of interest? Are you sure there is no faint band in the lane?
Basically what says in the title. My melting curves show 2 peaks, one at the expected Tm, another one around 10C lower. I thought the lower Tm would be primer dimer, so to check whether it was this or non-specific product I run my samples on a gel. Guess what, a beautiful single band of the expected size.
Any suggestions as to what that second peak could be????
Thanks in advance.
How was the dissociation curve in your negative controls?
If you have a more than several hundred bp product, and two regions which differ significantly in GC content, it is possible to have a single product exhibit multiple melting temperatures.
Hi phage, thanks for your answer I didn’t know that. The problem is some of my samples have 2 peaks, but others don't, when amplifying the same product. What is more, in the samples with 2 peaks, the second peak (lower Tm) is broader when the input cell number decreases.
I attach a power point slide trying to explain it. I'm not sure how much I'm actually allowed to show as I work in a company and they're quite tight about confidentiality.
I'm using an Ambion kit for viral RNA extraction, but my samples are actually mammalian cells. I think I have gDNA contamination in my samples, however the product size (228bp) is the same whether is from RNA or DNA, so I don’t think the 2 peaks are due to the genomic contamination. I'm rechecking all this with different primers now. However, I wonder if the DNA is having some effect as the cell number does affect the result.
Thanks so much for any help.
Hi phage, thanks for your answer I didn’t know that. The problem is some of my samples have 2 peaks, but others don't, when amplifying the same product. What is more, in the samples with 2 peaks, the second peak (lower Tm) is broader when the input cell number decreases.
I attach a power point slide trying to explain it. I'm not sure how much I'm actually allowed to show as I work in a company and they're quite tight about confidentiality.
I'm using an Ambion kit for viral RNA extraction, but my samples are actually mammalian cells. I think I have gDNA contamination in my samples, however the product size (228bp) is the same whether is from RNA or DNA, so I don’t think the 2 peaks are due to the genomic contamination. I'm rechecking all this with different primers now. However, I wonder if the DNA is having some effect as the cell number does affect the result.
Thanks so much for any help.
I have not used your system for DNA electrophoresis, but with a standard agarose gel you should be able to see the gDNA (just close to the well).
Generally, when a peak appears before the peak of your expected product, you have primer dimers. This hypothesis is reenforced by the fact that you get more of those when you have less material. You can always try a higher Tm. Or try another primer set, as you said 