Titration of Recombinant Adeno-Associated Virus (rAAV) Genome Copy Number Using Real-Time Quantitative Polymerase Chain Reaction (qPCR).

Abstract

This protocol is used to determine the concentration of DNase-resistant vector genomes (i.e., packaged in the capsid) in purified recombinant adeno-associated virus (rAAV) preparations. The protocol begins with treatment of the vector stock with DNase I to eliminate unencapsidated AAV DNA or contaminating plasmid DNA. This is followed by a heat treatment to heat-inactivate DNase I, to disrupt the viral capsid, and to release the packaged vector genomes for quantification by real-time polymerase chain reaction (PCR) using a set of standards (linearized plasmid used for vector production) containing known copy numbers. To accomplish high-throughput titration, the primer and probe sets used in real-time PCR are usually designed to target common elements present in most rAAV genomes, such as promoters and poly(A) signals. This strategy significantly reduces the number of PCRs, controls, and turnaround time. Several important controls should be included in the assay as follows: The first two controls should have a known copy number of the rAAV genome plasmid treated or not treated with DNase I. This control tests the effectiveness of DNase treatment. To control for potential cross-contamination between samples during the preparation process, a blank control containing nuclease-free water only should be processed and tested in parallel. A validation vector sample with a known titer should be included in every assay to monitor interassay variability. Finally, for the PCR run, a no-template control (NTC) is included to indicate cross-contamination during PCR setup.