I have a question concerning a commercial Taqman assay, which has been designed for the simultaneous detection of the target of interest (FAM-TAMRA probe) and an internal control (JOE-TAMRA probe). For the positive control, the manufacturer states that the Ct value must be lower than 36, which is the case (32) on our StepOne device (48 well plate, 25 uL per reaction). The internal control yields an average value of 24.5 in our experiments while manufacturer data, obtained with different types of samples, vary between 24 and 26. In our hands, the interassay CV is very low between experiments, both for the positive and internal controls.

The same samples have been tested on another cycler (LightCycler 480), yielding identical values for the internal control, but lower Ct values for the positive control (28). During this experiment, 10 uL/well was used (384 well plate). One could state that PCR efficiency is different between both devices, but this would result in different IC values too (or at least I suppose this). Am I correct? Importantly, our StepOne has no filter for the detection of TAMRA. Can this be an explanation for the different results?

What is the impact on the assay outcome if TAMRA fluorescence is not measured during PCR reaction?