I have validated a set of primers by dilution curve analysis using cDNA which expresses the gene.
I recently used these primers to look at the over-expression of this gene in a cell line that does not endogenously express this gene (FPKM = 0 in RNAseq). It worked well for all samples that were induced with my over-expression vector, but as a negative control, I included the non-induced cells as well. Here I get a CT of 32 but the melt curves look very bizarre with multiple peaks. I was expecting to obtain a CT of high 30s/40/undetermined. Also, when using two different amounts of cDNA for this sample (10 fold difference), I do not see the cycle number change in this sample where as it does for the induced samples. Wondering if I am nonspecifically amplifying something in this sample? Do you think this is real amplification? Is it a bad primer? Thanks