Melting curves for SYBRGreen

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Urska Sivka
Urska Sivka's picture
Melting curves for SYBRGreen

 Hello everybody!

I'm in dilemma about my melting curves so I ask you fine people to tell me if these are dimers/non specific products or not?

Melting curve A: ten fold dilutions series from 10^2 (blue) to 10^4 (green), final primer concentration:100 nM, cDNA was made from 2 ug total RNA, Ta=60 °C. 
Melting curve B: same dilution series as above 10^2 (blue) and 10^3 (red), 10^4 dilution serie show no ampliciation, primer final concentration 200 nM, same cDNA as above.
What bothers me about melting curve B are different peaks between replicates. Are these different PCR product? How is that possible if 10^3 is diluted from 10^2 and there all replicates have the same melting temperature? On the other hand difference in melting temperatures between replicates is only 0.3 °C. How big this difference (between melting temperatures) needs to be, to conclude there is nespecific amplification?

Thank you.

UrskaS

Ivan Delgado
Ivan Delgado's picture
Hi UrskaS,

Hi UrskaS,

If I understand your situation correctly, the only time when you see these different peaks is when you use primers at a 200 nM concentration. When the same primers are used at a 100 nM concentration all your melting curve peaks are ideantical. In my opinion you should simply assume that the different peaks detected when using primers at a 200 nM concentration are a result of an assay that has not been optimized, and instead simply use primers at a 100 nM concentration, which is the optimal primer concentration.

A difference of 0.3 oC is significant, but based on your situation it is hard to say if it means non-specific amplification. As mentioned above I would simply assume that your assay does not work well when you are using too much primer (200 nM) and instead use it with primers at a concentration of 100 nM. 

Best

 

Urska Sivka
Urska Sivka's picture
Hi Ivan,

Hi Ivan,

I'm sorry I wasn't clearer before. Melting curves are fo two different genes. What is bothering me in melting curve A is that "shoulder" before peak. Is that "shoulder" indicating non specfic product?
As for melting curve B, I don't understand how can one replicate have different melting peak than the other two (red lines)? It is also strange, because I did 10^3 dilution from 10^2 and in 10^2 dilution serie only one identical peak occurs in all three replicates. What I'm missing?

Regards,
UrskaS

Ivan Delgado
Ivan Delgado's picture
Hi UrskaS,

Hi UrskaS,
 
I see. Regarding the slight shoulder in peak A, I would not worry about it. It is not really a peak; its barely a bump. 
 
As for melting curve B, when the amount of DNA used to run the PCRs is too low, it is not unusual to see variation in the location of the peaks. As you can see the 10^3 peaks have a fluorescence that is half that of the 10^2 peaks. What this tells me is that this assay is not sensitive enough to reliably detect the amount of DNA present in the 10^3 dilution.
 
Hope this helps.
 

Urska Sivka
Urska Sivka's picture
Thank you Ivan, you just make

Thank you Ivan, you just make my day brighter ;)
About B case: how can I improve assay's sensitivity?

Regards,
UrskaS

Ivan Delgado
Ivan Delgado's picture
Hi UrskaS,

Hi UrskaS,

Anytime.

My approach to getting the best performing, and most sensitive, qPCR assay has always been to design at least two, ideally three, primer sets and run them all at the same time. More often than not one of those assay will work much better than the rest. I find that doing this saves a lot of time and effort instead of doing all the work necessary to optimize an assay that to begin with may not be ideal. In other words, spending a little more money in a few extra primers is much more cost effective that spending a lot of your time optimizing a single assay that in the end may not work. 

Happy holidays!
 

Urska Sivka
Urska Sivka's picture
Thank you again Ivan!

Thank you again Ivan!
I was also thinking about new primers.

Merry Christmas and successful New Year! UrskaS