Good day, I have a question related to particular results with qPCR. The performed assay has been fully validated but is now encountering some unexplained results. It's about the inconsistent appearance of the internal control. This does not always appear, or sometimes not at all, at the expected cq. As a result, the results cannot be officially recognised. We have already been busy troubleshooting but have not yet come up with a solution. We looked at possible inhibition, which does not take place. So this is not present. The template DNA is also present, so this is also excluded. We also performed a serial dilution series, with only the first dilution (cq21) emerging. At the same time, we are still far from the LOD. So we wonder if this is a primer/probe problem or something with the master mix. This is vortexed very briefly before use. If you have any idea where we should look further, these suggestions are most welcome! I really appreciate any help you can provide. We use; CFX opus (Bio-Rad) and Iq 5 multiplex power mix (Bio-Rad).
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