AAV Genomic Titer
AAV genomic titer refers to the concentration of viral particles containing vector genomes, typically expressed as genome copies per milliliter (GC/mL) or vector genomes per milliliter (VG/mL). However, this physical titer does not directly reflect AAV infectious titer, which can vary depending on the target cell type and may be affected by factors such as freeze-thaw cycles. While AAV genomic titer represents the theoretical maximum for infectious potential, it does not ensure actual transduction efficiency or biological activity.
Price and Turnaround
| QC | Target | Method | Turnaround | Price | Sample Requirement |
| AAV Genomic Titer | Not ITR | qPCR | 1-2 day | $50/sample | Primer sequences; ~20 μl samples |
| AAV Genome Titer | Not ITR | ddPCR | 3-5 day | $200/sample | Primer/probe sequences; ~20 μl samples |
Notes:
- AAV Packaging Services include the quantification of AAV titers through qPCR using gene-specific primers and the SYBR method. ITR titer analysis is also provided upon customer request.
- To ensure greater accuracy, an internal AAV reference is always included in the qPCR process.
- AAVnerGene offers a variety of commonly used primer/probe sets targeting sequences such as EGFP, CMV, WPRE, PolyA, and others (as shown below). Additional costs for probe synthesis may apply if a suitable primer/probe set is not readily available.
AAV References for PCR Based Titration
In the rapidly evolving field of gene therapy, the reliability and accuracy of research depend significantly on the use of high-quality reference materials. Both researchers and regulatory bodies like the FDA emphasize the importance of well-characterized AAV reference materials.
AAV References are essential for qPCR-based vector genome quantification. At AAVnerGene, All AAV References are produced using our unique and consistent AAVone production system. AAV References are available for a variety of serotypes, including AAV1, AAV2, AAV5, AAV6, AAV8, AAV9, AAVrh.10, and AAVrh.74. They all produced with our AAVone system, carrying a 2.8 kb of AAV-CMV-EGFP-WPRE-hGHpolyA genomes.
| Name | SKU | Price | Buy |
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qPCR vs ddPCR
AAV genome quantification is a critical step in gene therapy research and production, as it helps determine the concentration of viral genomes present in a sample. This quantification is essential for ensuring accurate dosing of AAV vectors in experimental and clinical applications. PCR-based methods are robust, easy, fast, and convenient.
Quantitative PCR (qPCR): qPCR is a widely used method for AAV genome quantification. It involves the amplification of a specific region of the AAV genome using AAV-specific primers. The amount of amplified DNA is measured in real-time, allowing for the quantification of the AAV genome copies in the sample.
Droplet Digital PCR (ddPCR): ddPCR is a variation of dPCR that partitions the sample into thousands of droplets. Each droplet acts as a separate reaction chamber, allowing for the absolute quantification of AAV genome copies. ddPCR offers high sensitivity and precision in AAV genome quantification.
| Feature | qPCR (Quantitative PCR) | ddPCR (Droplet Digital PCR) |
| Quantification | Relative quantification using standard curves | Absolute quantification without standard curves |
| Sensitivity | High, but less sensitive to small fold changes | Very high, detects small fold changes |
| Dynamic Range | Broad | Limited |
| Precision | Moderate | High |
| Throughput | High (up to 384-well format) | Moderate (up to 96-well format) |
| Cost | Lower, $50/Sample | Higher, $200/Sample |
| Applications | Gene expression, pathogen detection, etc. | Rare target detection, mutation analysis, etc. |
| Inhibitor Tolerance | Moderate | High |
| Ease of Use | Standardized and familiar | Requires more specialized equipment |
| AAV Reference | Yes | No Required |
ITR Titer VS Gene-specific Titer
ITR Titer: The commonly used AAV plasmid backbones, including our pAAVone, pAAVdual, and pAAVtri, carry two ITRs from AAV2. The wild-type AAV2 ITR is a 145nt hairpin structure. In the pAAV plasmids, the ITR has different versions, such as ITR145, ITR-I30, and ITR-119. Primers and probes targeting the common ITR region can be used universally for all AAV2 ITR-based vectors, which are included in the vast majority of vectors used in the field. This makes it a potential standardized assay for vg titrations. However, we have observed that viral genome titers obtained using the ITR qPCR are systematically higher (2-5 fold) compared to those obtained from qPCR assays utilizing with gene-specific primers/probes.
Gene-Specific Titer: Compared to ITR titer, the gene-specific titer is more accurate. However, primer design can significantly affect the results. qPCR efficiency can be influenced by the secondary structure of the AAV genome and primer annealing efficiencies. Since AAV preparations often contain incomplete genomes or partial genomes, the position of the primers can significantly affect the measured genome titer. While you can optimize primers for each individual sample, this reduces convenience and comparability, as each sample would be quantified with a different primer pair. Using primers targeting commonly used elements, such as the CMV promoter, WPRE, and PolyA, can yield relatively comparable results between vectors. Additionally, the precision of the assay can vary up to two-fold for the final titer value due to assay noise. Using a good AAV Reference in each experiment can reduce this noise by adjusting the results with an internal control.
Common qPCR primers/probes at AAVnerGene
Note:
We have more primers/probes set in stock. Please contact us (customer@aavnergene.com) to check whether the primers/probes are available or not for your AAV vectors.
| Target Gene | NO. | Label | Sequence | Test |
| ITR | P185 | qITR-F1 | CGGCCTCAGTGAGCGA | ddPCR, qPCR |
| P184 | qITR-R1 | GGAACCCCTAGTGATGGAGTT | ||
| P1192 | ITR-Probe | FAM-CACTCCCTCTCTGCGCGCTCG-TAMRA | ||
| CMV | P4528 | q CMV-F1 | GACGTCAATGGGTGGAGTATTT | qPCR |
| P4529 | q CMV-R1 | CGGACTTGGCATATGATACACT | ||
| EF1α | P948 | EF1a-F1 | TGCGAATCTGGTGGCACCTTCG | qPCR |
| P949 | EF1a-R1 | GTGCAGATCTTGGCCCGCATTTAC | ||
| HSYN | P240 | qhSyn1-F2 | TGCCTACCTGACGACCGA | qPCR |
| P241 | qhSyn1-R2 | CTCGCCGCATCCTGTTTC | ||
| CAG | P118 | qCAG-F | GACTGACCGCGTTACTCCCA | qPCR |
| P119 | qCAG-R | GCTTTCACGCAGCCACAGAA | ||
| GFP | P3105 | qEGFP-F | GAGCGCACCATCTTCTTCAA | ddPCR, qPCR |
| P3106 | qEGFP-R | TCCTTGAAGTCGATGCCCTT | ||
| P2654 | EGFP-Probe | FAM-ACAAGACCCGCGCCGAGGTG-TAMRA | ||
| eGFP | P4724 | q eGFP-F | AAGGGCATCGACTTCAAGG | ddPCR, qPCR |
| P4725 | q eGFP-R | TGCTTGTCGGCCATGATATAG | ||
| P4726 | q eGFP-probe | FAM-CTTGTGCCCCAGGATGTTGCC-BHQ1 | ||
| mcherry | p3921 | q-mcherry-F3 | AAGCTGAAGGTGACCAAGGG | qPCR |
| p3922 | q-mcherry-R3 | GCCGTCCTCGAAGTTCATCA | ||
| FLUC | P455 | qFluc-F | TGACCGAGAAGGAGATCGTG | qPCR |
| P456 | qFluc-R | GAGAATCTCGCGGATCTTGC | ||
| SaCas9 | P4721 | q saCas9-F | GCAACAAACTGAACGCCCAT | ddPCR, qPCR |
| P4722 | q saCas9-R | TCACGAACTTGTACACGCCA | ||
| P4723 | q saCas9-probe | FAM-CGTGAAGCTGTCCCTGAAGCCCTA-BHQ1 | ||
| WPRE | P116 | qWPRE-F | ATGAGGAGTTGTGGCCCGTT | qPCR |
| P117 | qWPRE-R | GCTGACAGGTGGTGGCAATG | ||
| WPRE3 | P3807 | qWPRE3-F | GCTATTGCTTCCCGTATGGC | qPCR |
| P3808 | qWPRE3-R | GGAATTGTCAGTGCCCAACA | ||
| hGH PolyA | P3962 | qhGH-f | TGGGTTCAAGCGATTCTCCT | qPCR |
| P3963 | qhGH-R | GTGAAACCCCGTCTCTACCA | ||
| BGH PolyA | P3189 | qbGH-F | GGAGTGGCACCTTCCA | ddPCR, qPCR |
| P3190 | qbGH-R | GCCAGCCATCTGTTGT | ||
| P5037 | qbGH-probe2 | FAM-TCCCCCGTGCCTTCCTTGACC-BHQ1 | ||
| SV40 poly A | P4768 | qSV40 poly(A)-F1 | CCAGACATGATAAGATACATTGATGAGTT | ddPCR, qPCR |
| P4769 | qSV40 poly(A)-R1 | AGCAATAGCATCACAAATTTCACAA | ||
| P4770 | qSV40 poly(A)-p1 | FAM-AGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTC-TAMRA |
Protocols of qPCR and ddPCR at AAVnerGene
- qPCR Protocol
- ddPCR Protocl