TITLE: AAV titration with qPCR
AUTHORS: Gerard Michael Coughlin

[DESCRIPTION]
Titration of AAV genomes in a purified sample is critical for ensuring accurate dosing. Titering AAV samples is typically accomplished by first treating samples with a DNase to degrade DNA not contained within the AAV capsid (i.e. unencapsidated DNA). Subsequently, the DNase is inactivated and encapsidated genomes are solubilized by denaturing and digesting the capsid. The number of DNase-resistant genomes in the treated sample(s) can then be absolutely determined using qPCR, with comparison to a standard of known concentrations. Alternatively, a droplet PCR method can be used, which bypasses the need for a standard. Here, we describe how to titer AAV samples using qPCR. 

[GUIDELINES]
This protocol is based on the titering protocol from Challis et al., Nat Protoc 14: 379, published in 2019. In the original protocol, 1.5 mL microcentrifuge tubes were used for all sample incubation and dilution steps, and for generating the standard DNA. This protocol presents a scaled-down version using 96-well plates, which saves on reagent and consumable costs and on plastic waste. We have not seen any systematic differences in titers obtained with this protocol vs. the previous protocol. 

[STEPS]
SECTION: Sample preparation
7. With a P10 pipette, place 2 µL of AAV sample into wells of a 96-well plate in triplicate. Load 2 µL of DPBS + 0.001% Pluronic F-68 into 3 more wells. Do not load samples into wells at the edge of the plate. Add 50 µL of DNase I solution (Step 5) to wells with AAV or DPBS + 0.001% Pluronic F-68. Surround the sample wells with 200 µL of UltraPure water, then seal plate and centrifuge briefly. Incubate at 37 °C for 60 min .

 

SECTION: Reagent preparation
2. Prepare DNase I buffer, by combining:

CaCl2: 55.5 mg 
1 M Tris-HCl: 2.5 mL 
MgCl2•6H2O: 508 mg 

With 250 mL of UltraPure water in a sterile bottle. Shake to mix, filter sterilize, and store at Room temperature for several months.

SECTION: Reagent preparation
3. Prepare Proteinase K buffer, by combining:

NaCl: 14.61 g 

With 250 mL of UltraPure water in a sterile bottle. Shake to mix until NaCl is dissolved. Add:

N-lauroylsarcosine sodium salt: 2.5 g 

And gently swirl to mix. Filter sterilize and store at Room temperature for several months. 

SECTION: Reagent preparation
4. Prepare DNA standard stock. Linearize a plasmid DNA containing the target sequence, using a single cutter restriction enzyme that does not cut within the PCR amplicon (e.g. something that cuts within the ampicillin resistance gene). Digest 20 µg of plasmid DNA with 60-80 U of enzyme, preferably in an overnight reaction to ensure complete digestion. Purify the reaction using two DNA clean-up columns, and measure the DNA concentration with a spectrophotometer. Dilute the DNA to 10 ng/µL , and measure the diluted DNA concentration in triplicate using a high sensitivity Qubit assay. Record the mean value and use for subsequent calculation. Pipette into 20 µL aliquots in PCR strip tubes and store at -20 °C .

SECTION: Sample preparation
6. Prepare DNase I solution. For every mL of DNase I digestion buffer needed, add 50 U of DNase I. Mix by inverting or by pipetting. Briefly spin down. 
SECTION: Sample preparation
5. Determine how much DNase I buffer (Step 1) and Proteinase K buffer (Step 2) you will need. Each AAV sample and a negative control (virus storage buffer: DPBS + 0.001 % Pluronic F-68) should be titered in technical triplicates. You will need 50 µL of DNase I buffer and 60 µL of Proteinase K buffer per well. For example, if you have 8 viruses to titer you will need to prepare at least:

(8 samples + 1 negative control) x 3 = 27 rxn

DNase I buffer: 27 rxn  x 50 µL/rxn = 1350 µL 
Proteinase K buffer: 27 rxn x 60 µL/rxn = 1620 µL 

Prepare extra to account for pipetting error and loading method. If using a repeater pipette or single channel pipette, 3 extra wells worth should be adequate. If using a multichannel pipette, 10 extra wells worth should be adequate. 

Pipette required volumes of DNase I buffer and Proteinase K buffer into clean tubes. 
SECTION: Sample preparation
8. After incubation, briefly centrifuge plate and carefully remove and discard the seal. Add 2.5 µL of 0.5M EDTA, pH 8 to each sample well, and mix by pipetting. Reseal plate with a clean seal, centrifuge briefly, and incubate at 70 °C for 10 min . 

 

SECTION: General
1. This protocol is comprised of 3 sections:

Reagent preparation (can be done months ahead of time)
Sample preparation (should be done the day of, or 1-2 days before running qPCR)
Sample and standard dilutions, and qPCR

The reagent preparation can be done on bench top. Sample preparation should be done in a Biosafety cabinet and in compliance with biosafety guidelines. We prepare sample and standard dilutions and qPCR reactions in a biosafety cabinet as well. 
SECTION: Sample preparation
9. Prepare proteinase K solution. For every mL of Proteinase K buffer needed, add 100 µg of Proteinase K. Mix by inverting or pipetting, and briefly spin down. 
SECTION: Sample preparation
10. Once 70 °C incubation is completed, allow plate to cool to room temperature, then centrifuge. Carefully remove and discard the seal. Add 60 µL of Proteinase K solution to each sample well. Reseal plate with a clean seal, centrifuge briefly, and incubate at 50 °C for 120 min to   . 
 

SECTION: Sample preparation
11. Inactivate the Proteinase K by incubating plate at 95 °C for 10 min. Following incubation, plate can be stored at 4 °C for a few days.
 

SECTION: Sample and standard dilution, and qPCR
13. Retrieve sample plate and centrifuge to collect liquid at bottom. Carefully remove and discard seal. Add 120 µL of UltraPure water to sample wells, and mix by pipetting. Set plate aside. 
 

SECTION: Sample and standard dilution, and qPCR
12. Before starting, create a "plate map" and determine the number of qPCR reactions you will run. In total three 96-well plates will be needed:

The sample plate: this is the plate that you have already prepared, containing the DNase I- and Proteinase K-treated AAV samples, as well as the negative control. 
The dilution plate: this plate will be used to create the standard dilution series and to further dilute the samples. As with the samples, each standard concentration is run in triplicate. 
The qPCR plate: this plate will be used to set up and run the qPCR reactions. 

The dilution plate and the qPCR plate will have the same layout. 24 wells will be used for the standard, which will occupy wells A1-B12. The processed samples will be added starting at well C1. Finally, a water control should also be used, loaded after the samples. 

SECTION: Sample and standard dilution, and qPCR
14. Prepare standard dilutions. In a new 96-well plate, add 5 µL of linearized standard to wells B10-B12. Then add 45 µL of UltraPure water to each well of rows A and B (24 wells total). When adding 45 µL of UltraPure water to wells B10-B12, mix well by pipetting. This will create a 1:10 dilution of the standard stock in wells B10-B12. 

Seal plate and centrifuge, then carefully remove and discard seal. Using 3 tips on a P10 multichannel pipette, prepare serial dilution of the standard. Working backwards, pipette 5 µL from B10-B12 into B7-B9 (i.e. the standard from B10 will be diluted in B7), mix by pipetting up and down 20 times to mix, then transfer 5 µL from wells B7-B9 into wells B4-B6, pipetting up and down 20 times to mix. Continue this process until wells A1-A3.

 

SECTION: Sample and standard dilution, and qPCR
15. Prepare sample dilutions. In the same 96-well plate as the standard, add 2 µL of DNase I-, Proteinase K-digested samples (Step 13) to the wells below the standard, starting at well C1. Add 198 µL of UltraPure water to the 2 µL and mix by pipetting. Seal and spin down the dilution plate. 

The sample plate can be sealed and stored at 4 °C in case it needs to be used again. 

 

SECTION: Sample and standard dilution, and qPCR
16. Set up the qPCR mastermix, according to manufacturer directions. Transfer 2 µL of diluted standards and samples (from dilution plate, Step 15) to qPCR plate, add master mix, and seal with an appropriate seal. Run the qPCR reaction according to manufacturer directions. 

 

SECTION: Sample and standard dilution, and qPCR
17. Analyze results using Supplementary Table 4 from Challis et al., Nature Protocols 14: 379.