dilution dependent melt temp variation
I'm new to the forum and have done quite a few qPCR experiments in my career. I am here today though, because I have never seen something like this before and don't know how to interpret.
I am doing a primer optimization for a Sybr green based qPCR experiment. Ran a dilution series of my cDNA with Rox as a reference dye and for detecting product. My primers are the same in each well. I was looking at my melt curve analysis with my run to make sure I was getting only a single product during the amplification. I have attached an image of it.
Each peak represents the average of technical duplicates in the dilution series. The thing that is particularly weird is that the melt temp appears to be dependent on the dilution in the series. So in the picture the far left, yellowish asterisk, peak is the 1 x10^-4 dilution, gray diamonds is 10^-3, green triangles is 10^-2, red squares is 10^-1, and blue circles is 10^-0. Each sample is made from the same starting cDNA. The amplification curves are good (as far as their shape and when they appear, mid-20s for the first sample). While it is not a perfect effeciency (approximately 140%, R-squared of .92) it didn't seem that major other products are being detected as their is a single point within each duplicate.
Has anyone ever seen anything like this? Any insight on this issue would be much appreciated. And if there is any other information that you need to help analyze, just let me know.