Hi Ethan,

from the curves I see here I would conclude that you have no amplification at all. Expecially, picture A shows no amplification exceeding the background noise.

In picture B it looks a little bit better, but I still would consider this as no real amplification.

If you use SybrGreen did you have a look at the melting curve to see if you have to see if you have a single amplicon (I am not sure what assay you are using as I can not really see this on the provided pictures)?

I would make a gel run with your qPCR products after finish the qPCR to see if you really amplifed something (for me it doen't look like that).

It also doesn't look like that you have a working positive control (maybe I just didn't see it) - make sure that you always include a working positive control, as well as a negative control.

Even if you had sucessfull amplification in your "normal" PCR you can have variations in the qPCR (e.g if you have different polymerase, different primer concentration, different magnesium concentration). So make sure that the PCR set up is identical to the setup for the qPCR when are testing your assay.

However, I would also have a look of your template integrity, because of the high background noise and the absence of amplification.

Especially, if you use RNA as a template, because RNA is really prone to degradation.

So if you are unlucky you can have amplification in your normal PCR, because the template is still ok but you might have difficulties when setting up your qPCR reaction.

Check your template quality on a gel, because checking it only with of photometer (what a lot of people do - I am not sure how you are doing it) will only tell you the concentration and if you have impurities and not if you have a a good template quality.

High background noise might be also caused by to high template concentrations in your qPCR reaction - this should also be avoided (however, I don't think this is the main problem here).

I hope this helps but maybe there is also additional input from other researchers as trouble shooting for qPCR is not an easy task :-)

Hi Ethan,

I just saw that I didn't not read your question properly and that you designed primer and probe sets for your qPCR. For this reason you can't do the melting curve analysis (at least it will make no sense) as you are obviously not using SybrGreen :-)

However, I still would recommend a gel run after the qPCR to see if you have amplified your desired amplicon and also if you may have the formation of primer dimers which - even if you are not getting false positives from primer dimers if you are not using SybrGReen - still can interfere with the efficiency of your PCR.

Double check your probe sequence to verify that your probe anneals properly. Maybe also check the sequence of your PCR product - sometimes annotated sequences can differ from your amplified sequence - depending on your template - and this could also interfere with probe binding.

Hey Ethan,

I recommend double-checking the concentrations of your primers and probe—both the stock and working solutions. Make sure to calculate the final concentration of each component in your master mix, regardless of what’s stated in the manufacturer’s datasheet. It would also be helpful to run a temperature gradient qPCR to determine the optimal annealing temperature (Tm) for your primers, and then repeat your PCR using that annealing temperature. Additionally, ensure the quality and integrity of your template DNA are good. Hope this helps!