As of January 2020 all lots of AAV produced at Addgene are titered using ddPCR. This change is effective for all AAV lot numbers above v69007. Lots previous to this were titered using qPCR. ddPCR is more precise and reproducible than qPCR based titration and, unlike qPCR which provides a relative quantification, provides an absolute count of the target DNA copies. Because ddPCR does not rely on a standard curve, titers are more accurate thereby improving consistency across lots.
Due to the accuracy and sensitivity of the assay, ddPCR titers tend to be approximately 2-fold higher than those measured via qPCR. For an explanation of why ddPCR titers are higher than qPCR please see the work of Lock et al. 2014. Importantly, and for the purpose of your experiments, please be advised that the potency of our viral lots has not changed. Consequently, you should not dilute your virus 2-fold if comparing a new ddPCR titered lot to an old qPCR titered lot of the same virus. For example, if you previously used 1x1011 GC of a qPCR’d lot of virus when reproducing the experiment you will need to use at least 2x1011 GC of a ddPCR’d lot. As always, blue capped vials have been produced by Upenn and were titered by ddPCR.
AAV titering is performed prior to freezing, and we have observed that physical titer values are not affected by freeze-thaw.
All lentivirus titering is performed on viral preparations that have been stored at -80°C and thawed on ice. This ensures that any loss of titer associated with the recipient's initial thaw will be accounted for in our reported titers. Target cells are infected with serial dilutions of the lentivirus and harvested several days later. Genomic DNA is extracted and integrated copies of RRE are measured via ddPCR.
For a complete explanation of our viral service titering protocols, see our Viral Production page.