I am new to QPCR. However, from what I do know I do not think our approach is appropriate. Our assay is a ligation assay in which the ligation probes have incorporated universal PCR primers. So, for all 50 targets the sequences for the PCR primers are identical and the sequences for the ligation probes (left and right) are different. The sizes are the same so after ligation to the target the resulting amplicon is 148 bp long. The problem is that we are QC testing our probes (ligation probes) by determining the Ct values at two concentrations of synthetic target mimic (10 pM and 1 pM , and negative) and making the assumption that the Ct values should be identical/ similar for all 50 targets since they are not long targets (the sites only bind to the ligation region of the ligation probes) and that they are not complex like RNA. The probes were designed by a bioinformatician. The issues are two fold: 1. they are not similar and 2. the difference in Ct values between the different concentrations vary significantly with some giving an appropriate 3.43 and others giving 1.85 or 5.9.