qPCR Adeno-Associated Virus
Adeno-associated virus (AAV) is currently the most popular gene delivery vector for in vivo gene therapy. However, variability in titration methods between different laboratories affects the reproducibility of experiments and the evaluation of safety and efficacy in clinical trials. We describe an optimized protocol for AAV titration, which includes standard preparation of quantitative PCR (qPCR) and quantification and treatment of AAV samples prior to titration of qPCR and digital droplet PCR (ddPCR).
During the development of the protocol, we observed that the quantification of the qPCR standard depended on its conformation and that the quantification based on A260 overestimated the plasmid copy numbers, introducing a significant error. The linearized free inverted terminal repeat (free ITR) and supercoiled standards were compared to the Enhanced Green Fluorescent Protein (EGFP), SV40p (A), and AAV2-ITR qPCR assays and we found that using the AAV2-ITR assay in conjunction with El Linearized or supercoiled standard led to an overestimation of titers, whereas the EGFP and SV40p (A) assays were more accurate with the linearized standard.
Finally, we compared the extraction of AAV1, AAV2, AAV5, AAV6, AAV8, and AAV9 genomes by thermal denaturation, proteinase K treatment, and kit extraction. Extraction of the kit, which contained proteinase K treatment in denaturing buffer before spin column purification, significantly increased the acquired titers for all serotypes in both qPCR and ddPCR. These improvements resulted in inaccurate quantification of the ATCC reference standard and a robust and reliable protocol for AAV titration.
Cat # + Size
An ideal tool for quantification of the adeno-associated virus by qPCR
Quantification kit based on qPCR
• An ideal tool to quantify adeno-associated virus
Features and benefits
- High specificity and sensitivity
- Non-specific minimum fund
- Results ready in less than 2 h
- Reliable and ready to use
- Virus lysis buffer
- Reagent mix
- STD1 (AAV 1 standard)
- STD2 (AAV standard 2)
- DNase reaction mix
For research use only! It is not for use in humans.