I'm new to qPCR, and I’ve been testing the AR expression on canine tissues. I’m currently using a pair of primers that have been tested in other article for canines.The issue I’m having is that I have very early amplification of ntc AR (before the amplification of cDNA templates) but also this amplification curve looks weird since it reaches quickly the plateau phase (flattened). This problem persisted even after changing to a much more specific cycling. If anyone has any tips, or ideas on how I may be able to explain this, I’ll be very gratefull.
The cause of the signal in the no template control will be contamination. It could be that the reason for the different plateau positions is the presence of reaction inhibitors, depletion of reagents, or possibly different binding efficiencies between template (and contaminating DNA) and the primers. I'm not sure about that last one but it kinda makes sense in my head: any contaminating DNA that binds to the primers probably won't bind as efficiently as the template that the primers have been designed for so the reaction is inhibited by accumulation of dsDNA faster.
Either way the cause is going to be excessive contamination. Try using more template in your samples (increasing you signal to "noise" ratio), or be more careful when preparing your samples.
There are extraordinary measures that can be taken to remove contaminating DNA (e.g. treating the water with DNase and then deactivating it) but these often aren't necessary and the added steps can actually increase contamination, it's usually best to keep it simple.