Dear Mark

I think you are using very low cDNA dilution.

You have to increase the cDNA concentration because it is not picked up to a threshold value. I recommend to set up a experiment with different cDNA concentration. and you can also increase the extension time.

All the best

Thanks Meenakshi

The assay does not include a reverse transcription step for cDNA, it is amplifying genomic DNA. It is primarily a diagnostic assay which is then used for genotyping so I don't want to change the protocol too much.

Hi Mark, 

This looks like some kind of instrument problem to me. Most real-time PCR instruments have algorithms that correct for background fluorescence, among other things. In your case, the background levels seem to vary a lot and the instrument may not be compensating for them correctly. 

Try looking at the unprocessed raw data from each individual filter channel. I think you'll find a fine-looking amplification curve hiding there. 

If not, then the fluorescence level is rising and then falling again (ie your amplification curve is showing what is really going on), in which case something very strange is going on. It's worth taking a look at your samples to see whether they're turning yellow or evaporating or throwing a precipitate or something like that.