I am trying to design a real-time PCR assay for copy number determination of an antibody gene in mammalian cells, using TaqMan fluorescent probes.
The issue is, I need to find a way to improve the sensitivity of the assay. So far, it works perfectly in the range 108 – 104 copies of either antibody chain per reaction (acceptable efficiency, Pearson correlation, amplification is every three cycles). When 10-103 standards are added to the standard curve, however, they all cross the threshold line at about 24 cycles along with the 104 standard, irrespectively of the fact there is far less DNA in them, and they should cross the line a lot later.
So, far, we have tried optimising primer concentrations and cycling conditions, tried different primers, ran the assay on a different machine…. The problem still exists.
Any bright ideas or suggestions on what to try next?