Quantification of Adeno-Associated Virus with Safe Nucleic Acid Dyes
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Abstract
Adeno-associated virus (AAV) is the most commonly used viral vector for both biological and gene therapeutic applications. Although many methods have been developed to measure quantity attributes of AAV, they are often technically challenging and time-consuming. Here, we report a method to titer AAV with GelGreen ® dye, a safe green fluorescence nucleic acid dye recently engineered by Biotium company (Fremont, CA). This method, hereinafter referred to as GelGreen method, provides a fast (∼30 min) and reliable strategy for AAV titration. To validate GelGreen method, we measured genome titer of an AAV reference material AAV8RSM and compared our titration results with those determined by Reference Material Working Group (ARMWG). We showed that GelGreen results and capsid enzyme-linked immunosorbent assay results are comparable with each other. We also showed that GelRed ® dye, a red fluorescence dye from Biotium, can be used to directly “visualize” AAV genome titer on a conventional gel imager, presenting an especially direct approach to estimate viral quantity. Finally, we showed that GelGreen and GelRed dyes can also be used to quantify self-complementary AAV (scAAV) and crudely purified AAV samples. In summary, we described a technique to titer AAV by using new generation of safe DNA dyes. This technique is simple, safe, reliable, and cost efficient. It has potential to be broadly applied for quantifying and normalizing AAV viral vectors.
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DyeBA genome titer for an rAAV reference material differed significantly from qPCR and agreed with capsid titers obtained by ELISA.
“However, the genome titer found with this method for an rAAV reference material differed significantly from results obtained with qPCR and instead agreed with capsid titers obtained by ELISA.”
From Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies (2021) · cites this paper as [59]
DyeBAs can directly quantify genome titer from at least crudely purified samples using FS signals.
“Direct quantification of the genome titer from at least crudely purified samples is possible using FS signals from dye-based binding assays (DyeBAs).”
From Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies (2021) · cites this paper as [59]
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- Authors
- Xu, J., DeVries, S. H., & Zhu, Y.
- Affiliations
- Northwestern University (US)
- Corresponding author
- Zhu, Yongling
- Venue
- Human Gene Therapy
- Publisher
- SAGE Publications
- Published
- 2020-10
- Volume
- 31
- Issue
- 19-20
- Pages
- 1086-1099
- ISSN
- 1043-0342 (ISSN-L)
- 1557-7422
- PMC
- PMC7588323
- OpenAlex
- W3021961135
- Cited by
- 25
- References
- 47
- Citations by year
- 2026: 5 · 2025: 5 · 2024: 6 · 2023: 1 · 2022: 4 · 2021: 4
- FWCI
- 1.32
- Type
- journal article
- Language
- en
- Open access
- green
- doi.org
- Locations
- Human Gene Therapy (publishedVersion)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Northwestern University
- National Eye Institute (R01EY018204, R01EY030169)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Molecular Biology Techniques and Applications
- Keywords
- adeno-associated virus, self-complementary AAV
- MeSH terms
- Dependovirus, DNA, Viral, Fluorescent Dyes, Genetic Vectors, Humans
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