Dear Yaghma,

melt peaks below 75 degrees can be caused by primer dimer amplification. PCR products, unless they are very short or A/T rich would normally be above 75 degrees.

Have you put your PCR products on an agarose gel to check what size they are? It sounds like you may not be amplifying anything except primer dimers (or possibly some contamination) in either sample or NTC.

One point is that you say you are using 10 uM primer concentration - is this the final concentration in your PCR mix or is this the stock concentration of your primers? Just to clarify what I mean: do you use 1 ul of your 10 uM primers in 10 ul PCR reaction?Then the final concentration is 1 uM.

Or do you use 1 ul of a 100 uM primers in 10 ul PCR? Then the final concentration is 10 uM.

I ask because 10 uM is a very high primer concentration and would very likely lead to primer dimers if there is even just a little bit of homology.

The other point is that if you do more than 35 or 40 cycles (depending on your PCR setup) quite often there will be some sort of amplification in the NTC. This is simply due to the fact that the conditions of PCR may encourage annealing of even small stretches of homology and subsequent amplification if there is no fully homologous template available that would be a preferred partner for the primers. If there is at least ~6.6 Cq (Ct) difference between the NTC and your samples you can still use the data, because the possible contribution of the nonspecific amplification is

Dear Masood

You mean no amplification curve at all !! I am afraid you have mild contamination in your master mix which you are unable to see in amplification plot. Try to change the threshold to 0 and look for amplification plots.

Good luck!

1. sometimes automatic threshold value and baseline cycle may affect your amplification curve,you need to set it manually

2. what is the "product melt curve"you mentioned? You sure it is the expected peak? Melt peaks below 75  celcius will need more confirmation

At 0.03 threshhold value there is no NTC amplification curve, but I find the melt peak below 75 celcius for NTC.  For rest of the samples I am getting nearly the same melt peak and amplification curves with Ct values between 29-33.

Dear Yaghma,

melt peaks below 75 degrees can be caused by primer dimer amplification. PCR products, unless they are very short or A/T rich would normally be above 75 degrees.

Have you put your PCR products on an agarose gel to check what size they are? It sounds like you may not be amplifying anything except primer dimers (or possibly some contamination) in either sample or NTC.

One point is that you say you are using 10 uM primer concentration - is this the final concentration in your PCR mix or is this the stock concentration of your primers? Just to clarify what I mean: do you use 1 ul of your 10 uM primers in 10 ul PCR reaction?Then the final concentration is 1 uM.

Or do you use 1 ul of a 100 uM primers in 10 ul PCR? Then the final concentration is 10 uM.

I ask because 10 uM is a very high primer concentration and would very likely lead to primer dimers if there is even just a little bit of homology.

The other point is that if you do more than 35 or 40 cycles (depending on your PCR setup) quite often there will be some sort of amplification in the NTC. This is simply due to the fact that the conditions of PCR may encourage annealing of even small stretches of homology and subsequent amplification if there is no fully homologous template available that would be a preferred partner for the primers. If there is at least ~6.6 Cq (Ct) difference between the NTC and your samples you can still use the data, because the possible contribution of the nonspecific amplification is

Dear Petra Thanks for your reply.

 1) I am working on miRNA. Do you think melt peak of the product below 75 is possible? 

2) I am using 0.4 ul of 10uM of the stock concentration for each primer in 0.1 ml PCR tube in (10 ul final volume).

3) Each time I am doing 36 cycles.

4) Please some literature that can help me to learn more about this topic.

You should check Applied Biosystems website they have a lot of good hints on troubleshooting. Considering your remarks I agree with Petra. You probably have a primer dimmer or a very mild contamination. You should try diluting a new primer stock and also run your primer sequence on IDT Oligo Analyzer. The online program will show you all possible primer dimmers, hairpins and the DeltaG for each one of them. 

HI Pranay, Chen, Petra and Andre

Many thanks for the replies. Really appreciate it.