For comparing the quality of two products, I used the same sample and primers to do qPCR, the results are as follows. As the figure shows, Tm values are a little different. I wonder if this can tell which of the two products is better, or whether it is judged by other parameters? Thank you.
Hi,
Tm can vary with different buffers due to difference in K+/ salt concentration and also change in MgCl2 can shift Tm values.
Did you use same mastermix in both the runs?
If you amplify typical short sequences, how do you define "quality" on these amplicons (what should be the difference between a "high-quality" and a "low-quality" amplicon)? I don't think that this is a sensible question to adress. Most likely, the Tm differences are results of different treatments or sample-sources that cause slight differences in the buffer conditions.
If you want so check RNA/DNA degradation, better amplify long sequences and measure the Ct. The more degraded the NA is, the higher the Ct.
The Tm of a melt curve is the least informative part of the experiment. They can shift due to what type of detection you chose (SYBRGreen, EvaGreen, etc.), buffer type, and other non-consequential factors.
If you are really curious, run out your samples on a gel to take a look at them.
The best product is what ever primers and conditions give a 95%-105% efficiency rating. That's the standard for publication.
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