When I compared the melting curve I got for a qPCR (SYBR green) run with that of another run done a few weeks earlier using the same primer set and identical cycling conditions, I find that the peaks were at different temperatures (78 C for the first and 84 C for the second). In both the melting curves, there was only a single peak and no primer dimers. The samples (cDNA) used were different between the runs but in both cases were isolated from the mouse liver. Why could this be happening?
Hello, Joe.
first of all, as is normal in experimental evaluations, if you are excluding the possibility of varables EXCEPT the different cDNA samples, you should point your attention to this simple difference. maybe some little variation in RT efficiency, or in the starting RNA quality, can easily affect the reproducibility.
As you know, cDNA templates sometimes show rapid impoverishment in quality and integrity, even with the passing of freezing-thawing cycles; and different synthesized cDNAs have unexpected different sensitivity to degrading troubles. more than a few times, differential quality of cDNA (RNA) simply explains different melting curves, temperatures, peaks etc...
Hi Joe,
as Amilcare said the differences in your cDNA and starting RNA samples could be the main causes of different melting peaks. You can try to check the quality of starting RNA by bioanalyzer (RIN>10), and performing if possible RT step in a single run, in the same day with the same RT kit for all the samples, in order to reduce confounding variance. You can also check if your Sybr-green assay is targeted on a specific gene/isoform (design on specific splicing junction for example) or could include different splicing variant of the same transcript. In that case then is quite normal to find different melting peaks among samples or, even worse, multiple melting peaks on single sample.
Hi, Joe
Did you calculate the melting T of your PCR profuct? Most of the programs for primer design allow to perform this evaluation. +/- 3 degrees of deviation is possible from the designed T, but not as much as in your case. Additionally there are some possible explanations: 1) one of the picks is not your product. 2) you have got a splice variant.
Thanks Amilcare, Alessandro, Vladimir and Andrey. I did some more investigation into my recent qPCR results and I've found that the melting curve Tm has been the same in the last few qPCRs I've done recently with each of my primer sets. Interestingly, there is a difference of 3 to 5 C in the Tm for every primer set between runs done in the last couple of weeks versus those done before a month. The PCR machine was serviced some time during this time. I wonder if this may be responsible for the differences seen...
Hi Joe, Another way to confirm your product is by using DNA sequence the pcr Product or run it on a gel to confirm single band and product size. Usually, +or- 4-5 C is okay as long as you get a single peak. Moreover, melting curve is not used for diagnosis, it is an indicator. Hope this helps you
I would definitely run your recent products on a gel to make sure that they match in size with what the product size was when you originally ran the assay. Hopefully, you checked it on a gel then, but you could also just look at what the predicted size of the PCR product is with these primers to make sure it matches.
Another important factor is the source of the Sybr Green mix. Different commercial mixes have different salt concentrations and other additives which can radically affect the melting temperature of the primers and the PCR products. I have especially noticed this when I have been comparing different commercial Sybr green mixes. If you have changed Sybr green mixes since your earlier runs, this could be a likely cause of the apparent difference.
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