End-to-end High Throughput Quantitative PCR-based rAAV Genome Titering Workflow

Introduction

Authors: Paulina Kanigowska, Ph.D., Elizabeth Gendreau, Kevin Hong, Alicia Byrn, Lexie Cui, Ph.D., Chris Bremner, Henri-Louis Girard, Ph.D., Tasnia Chowdhury

Quantitative PCR (qPCR) is a gold-standard DNA quantification method, which uses PCR DNA amplification in the presence of either a fluorescent DNA intercalating dye or a fluorescent DNA hybridization probe to measure an unknown DNA concentration in a reaction. It is a versatile laboratory technique, which can be further adapted to e.g. quantification of miscellaneous RNA species, gene expression profiling or genotyping.

Here, we present how Ginkgo leveraged qPCR to quantify the genome titer of recombinant adeno-associated virus (rAAV) particles. We describe how we developed a fully automated, qPCR-based rAAV genomic titering workflow on our RAC platform, including upstream pre-treatment of rAAVs (including rAAVs in crude mammalian cell lysate and purified rAAVs) and downstream data analysis to maximize human hands-off time. We further show how our approach enabled workflow runs up to ~23 hrs long and end-to-end processing of up to ~7,500 samples, without any human intervention, and how it ultimately helped Ginkgo’s Operations Team quantify DNA concentrations of >100,000 samples to date.

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