The AAV titration protocol can be used to determine the number of genome-containing particles in an AAV products with qPCR method (Sybr Green). This protocol is suitable for 96/384-well plates with a reaction volume of 10 µL.

Equipment

• Applied Biosystems qPCR instrument
• BIO-RAD incubator

Reagents

• DNase I
• EDTA
• Proteinase K
• Universal SYBR master mix
• 2XRox dye
• Nuclease-free water
• Primer pair targeting specific gene
• Specific gene-containing plasmid
• Restriction enzymes
• Internal AAV reference [AAVnerGene, SKU:DJ001001-AAV9]  

Preparation

• DNase I solution: 500 μL DNase I buffer with 5 μL DNase I  (DNase I: 200 μg/ml*0.1ml=20μg, DNase I buffer: 10 mM Tris-HCl, pH8.0, 10 mM MgCl2, 2 mM CaCl2, 0.1% Pluronic-F68)

• EDTA solution: 0.45 M EDTA

• Proteinase K solution: 500 μL Proteinase K buffer with 10 μL Proteinase K (Proteinase K: 200 ug/ml -NEB/https://www.apexbt.com/proteinase-k.html, Proteinase K solution: 1 M NaCl, 0.1% Sarkosyl)

• Standard stock solution: Prepare a linearized plasmid stock of 1×10⁹ molecules/μL, aliquot into tubes and store at -80℃. (https://www.thermofisher.com/us/en/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/thermo-scientific-web-tools/dna-copy-number-calculator.html)

• Internal AAV reference [AAVnerGene, SKU:DJ001001-AAV9]:https://sabrinahe22de09a181.wpcomstaging.com/product/aav9-reference/

*Tips: When linearizing the standard plasmid, ensure that the resulting fragment closely resembles the genome of the AAV product being tested. Using restriction enzymes that cut near the ITRs will provide the best mimicry of AAV genomes, resulting in a more accurate representation during analysis.

Digestion

Treat AAV samples and AAV reference with DNase I to remove any residual plasmid DNA from the production process. Then, use Proteinase K to degrade the viral capsid and release the encapsulated genome for further analysis.

• Add 90 μL DNase I solution to 10 μL AAV sample, 37℃ for 60 min
• Add 6 μL EDTA solution (to stop DNase I activity), 20℃ for 10 min and then 75℃ for 20 min (to inactivate DNase I activity)
• Add 94 μL Proteinase K solution, 55℃ for 60 min and 95℃for 20 min

*Tips*: We highly recommend using 250 μL strip tubes and incubator to run these temperature program. If the next step will not be carried out soon, it would be better to store the digested sample at -80℃.

 

Dilution

Standard curve: Dilute the linearized plasmid standard with nuclease-free water stepwise, from 1e10⁷ to 1e10⁴ copies/μL.

Volume of 1×109 stock or previous dilution	Volume of nuclease-free water	Molecules per μL
10 μL of 1×109 dilution	90 μL	1×108
10 μL of 1×108 dilution	90 μL	1×107
10 μL of 1×107 dilution	90 μL	1×106
10 of 1×106 dilution	90	1×105
10 of 1×105 dilution	90	1×104

Sample dilution: Dilute the digested samples according to the dilution scheme in the table below.

Dilution Series	Volume of sample(μL)	Volume of nuclease free water (μL)	Dilution factor	Total dilution
Dilution 1 (Digestion step)	10 μL AAV stock	190 μL	20x	20x
Dilution 2	8 μL Dil. 1	192 μL	25x	500x
Dilution 3	10 μL Dil. 2	90 μL	10x	5000x

Note: At AAVnerGene, the titers of AAV samples typically range from 1×10¹² GC/mL to 1×10¹⁴ GC/mL. We use dilutions of 2 and 3, along with an internal AAV reference virus at 2×10¹² GC/mL. For crude AAV samples, titers usually range from 1×10¹⁰ to 5×10¹² GC/mL. Please adjust the dilutions accordingly based on these ranges.

*Tips*: Ensuring the quality of the sample dilution series is crucial. Each dilution should be mixed thoroughly by pipetting up and down at least 10 times, using at least half of the final volume (e.g., mix with more than 50 µL if the well contains 100 µL). Use a multichannel pipette to load both the standards and samples onto the qPCR plate for consistent and accurate results.

Set up and load plate

Mix thoroughly the reaction mixture according to table.

PCR mixture	1 well	96 wells
2x SYBR Green mixture, μL	5	480
50x Rox, μL	0.2	19.2
100 µM Forward primer, μL	0.025	2.4
100 µM Reverse primer, μL	0.025	2.4

• Add 5 µl PCR mixture into each well.
• Add 5 µL of diluted AAV samples and plasmid standards into each well.
• Seal the plate with a corresponding film.
• Spin down the sealed plate at 1,000 rpm, 1 min.
• Make sure the sample is at the bottom of each well and there are no significant volume differences
• Run the following protocol in your qPCR instrument using SYBR detection.

*Tips: There are two types of ROX dye: I and II. Please be sure to use according to the reagent compatibility guidelines.

Example of 96-well-plate set-up

Dilution

500x

5000x

500x

5000x

500x

5000x

1

2

3

4

5

6

7

8

9

10

11

12

A

S1

S1

S1

S1

S9

S9

S9

S9

S 17

S17

S 17

S17

B

S2

S2

S2

S2

S10

S10

S10

S10

S18

S18

S18

S18

C

S3

S3

S3

S3

S11

S11

S11

S11

S19

S19

S19

S19

D

S4

S4

S4

S4

S12

S12

S12

S12

S20

S20

S20

S20

E

S5

S5

S5

S5

S13

S13

S13

S13

Ref

Ref

Ref

Ref

F

S6

S6

S6

S6

S14

S14

S14

S14

Neg

Neg

Neg

Neg

G

S7

S7

S7

S7

S15

S15

S15

S15

STD-7

STD-6

STD-5

STD-4

H

S8

S8

S8

S8

S16

S16

S16

S16

STD-7

STD-6

STD-5

STD-4

Data analysis

*Final=power (10, Log) *20(Digestion)*500(Dilution)*1000(vg/μL→ vg/ml) *2(dsDNA)

* Typically, we consider the reference sample as a positive control. It is used to assess the stability of the test and correct for possible experimental errors, especially when preparing new linearized plasmid standards or testing under different detection systems.

• • Efficiency= 10^(-1/slope)-1.

• • Standard curve: R2(coefficient of correlation) ~ 1.0, E (efficiency of PCR) ~100% (90%-110%)

• • Melt curve analysis: a single peak should be seen. The presence of a second peak at a temperature of ~70-75℃ usually indicates the presence of primer dimers which can increase background signal and alter the Ct values of your samples.

• • Quality of duplicates: Exclude duplicates from analyses if there is more than a 0.5 Ct difference between them.