I am testing the residual plasmid quantity using qPCR in AAV gene therapy products.
Dear Feng Wang
I have personally not used PF68 for suspending viral particles so based on what I understood from your question here are my thoughts:
Could it be that the primers are you using to detect the residual plasmid DNA is also amplifying the host genome and therefore your primer detects residual DNA + AAV DNA + host DNA (cell line used to produce AAVs) ?
In case you already have a water control in the qPCR you can check if the water / any other reagent of the master mix is contaminated by any chance which might be giving you unexpected results. (Assuming that PF68 doesnot interfere with primer efficiency in qPCRs)
Your calculations seem fine to me. Have you tried simply repeating the qPCR with a fresh dilution series once more just to see if something went wrong the first time?
Shambhabi Chatterjee Thank you so much for your input!
1. I am running TaqMan, and the primers/probe work specifically. This was verified.
2. I repeated the assay 4 times and only one of them showed "Dilution = 2x Dilution 3".
The last step is 1:1 dilution, it is hard to be wrong with 1:1 dilution. That is why it is hard to figure out. The standards look well, PCR efficacy is ~955, R^2 is 0.9998.
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