Hello,

Context: I am trying to purify and quantify my AAV containing my plasmid of interest. I used HekAAV cells from Takara and transfected the cells with my plasmid of interest, pRepcap and pHelper. I extracted and purified my AAVs with the Takara midi kit. I ran an 8% SDS gel and was able to visualize VP3s and feint VP1 proteins.

Problem: In order to quantify the titer of the AAV, I treat my samples with DNase and proteinase K and run a qPCR with primers for the ITR of the AAV. My standard shows expected Cq increase with higher folds of dilution but for the same folds of dilution, my AAVs show the same Cq values. I repeated and observed similar Cq value with no change with change in dilutions.

Assumptions: The AAV are not well transfected and only the capsids are expressed. Both the plasmid of interest and pHelper were not transfected well. So, I only see VPs on SDS gel but no change in Cq values in PCR.

Looking for advice or suggestions as to what may have happened and how I can improve my titer quantification data.