I'm struggling with understanding and troubleshooting qPCR issues with cycles 1-3.
The rate of change for the cycles 1-3 is so high, it is tricking the software into thinking that is where the fastest rate of change is and throwing an error. See attached pictures of the PCR curve (Top) and rate of change graph (bottom).
My questions are as follows:
What are the factors and phenomenon that control PCR fluorescence in ~cycles 1-3? How do I eliminate the sudden increase at the start? Is there a hardware problem?
Note: Multiplex QPCR reaction using Taqman probes. Established and FDA approved assay.