If anyone has experience in qPCR I would be glad if you could read this and offer any advice.
Basically I have a running qPCR assay which is run as a duplex of target gene and housekeeping gene with Taqman universal mastermix on an ABI9700T.
I am currenlty trying to qualify the assay for use and I have hit a brick wall. I have mock samples spiked with a known copy number of my target. The target is an antobody as as such both the heavy and light chain genes are on one vector therefore if you assay with the heavy chain primer/probe set you should get the same copy number as assaying with the light chain primer/probe set.
The heavy chain/housekeeping gene duplex assay works and provides the expeced answer but the light chain/housekeeping gene does not, it is way under and at the lower copy numbders it does not detect anything.
My reagents are all fresh and probes are all single use aliquots, the primers/probes are designed with similar Tm, correct amplicon length etc etc.
I dont think this an assay issue I think it is specific to the gene I am targetting. i have sequenced the gene and it is fine. I have tried 6 primer/probe sets now.
I validate my primer/probe sets by runnng a standard curve against plasmid then the same in a mock duplex assay to check for specificity in the presence of the other reaction components, of the 6 primer/probe sets I tested.
The assays pass acceptance criteria of slope and R^2 then the test samples ar enot as expected.
Any advice or suggestions or if you have seen a similar thing would be useful, i have run out of ideas now.