ddPCR Titration of AAV Vectors

This protocol describes how to use droplet digital PCR (ddPCR) to titer purified recombinant Adeno-associated viral vectors (AAV). This protocol specifically uses primers and probes targeting the ITR elements in the viral vectors but can be modified for other targets. The dilution series outlined in this protocol are based on an AAV titer range of 5E+12–5E+13 GC/mL, where GC is genome copies and generally represents the physical number of viral particles. Users may need to run lower or higher dilutions depending on their particular sample. For additional tips on AAV titering using ddPCR, read this blog post on ddPCR for AAV quantification.

This protocol was modified from the publication Lock M, Alvira MR, Chen SJ, Wilson JM, 2014.

Before Starting

• To reduce the risk of contaminating reagents we recommend making small aliquots of master mixes, primers and probes prior to use.
• Thaw the master mix, primers, and probe on ice before use.
• Wipe down all pipettes and surfaces with 10% bleach.

Safety Warnings

AAV is generally considered biosafety level 1 but may require BSL-2 handling depending on the insert. Please ensure that you are in compliance with your institution’s biosafety regulations.

Last Update: June 12, 2023

• Class II, Type A2 Biological Safety Cabinet, Labconco 302411100
• Droplet Digital PCR System, Bio-Rad, DX200
• Thermal Cycler, Bio-Rad, T100
• PCR Plate Sealer, Bio-Rad, PX1
• 1-10 µL single channel pipette
• 20-200 µL single channel pipette
• 200-1000 µL single channel pipette
• 1-10 µL multichannel pipette
• 2-50 µL multichannel pipette
• 20-200 µL multichannel pipette
• Vortex, VWR, 10153-688
• Mini Centrifuge, Thermo Scientific, 10199-452
• Ice bucket
• 96-well freezer blocks (x3)

• Molecular Biology Grade Water, Hyclone, SH30538.02
• GeneAmp 10X PCR Buffer, Applied Biosystems, N8080129
• ddPCR Supermix for Probes no dUTP, Bio-Rad,1863023
• 10% Pluronic F-68, Thermo Fisher, 24040032
• Droplet generation oil, Bio-Rad, 1863005
• DG8 cartridge, Bio-Rad, 1864008
• DG8 gasket, Bio-Rad, 1863009
• DG8 cartridge holder, Bio-Rad, 1863051
• 8-strip PCR tubes, Axygen, PCR-02-FCP-C
• ddPCR 96-well PCR plates, Bio-Rad, 12001925
• 48-well dilution plate, Bio-Rad, MLL4801
• Pierceable Foil Heat Seal, Bio-Rad, 1814040
• Microseal adhesive seal, Bio-Rad, MSB1001
• Polystyrene Reservoirs, VWR, 89094-662
• Microcentrifuge tubes, VWR, 87003-294
• Primers/probe targeting ITR:
• ITR Forward Primer: 5’-CGGCCTCAGTGAGCGA
• ITR Reverse Primer: 5’-GGAACCCCTAGTGATGGAGTT
• ITR Probe: -FAM-CACTCCCTCTCTGCGCGCTCG-BBQ-

Preparation

1. Before handling any viruses get materials ready.
2. Ensure that primers, probe, 10X PCR buffer, and master mix are thawed.
3. Vortex primers, probe and master mix for 15 seconds then spin 10 seconds in a mini centrifuge and place on ice.
4. Wipe down a DG8 cartridge holder with bleach and place in the Biological Safety Cabinet (BSC).
5. Make sure that the BSC to be used for dilution and the BSC to be used for droplet generation are supplied with sufficient pipette tips and reagent reservoirs.

*Pro-Tip* Prepare the viral dilutions in a separate biological safety cabinet (dilution BSC) than that used for master mix preparation and droplet generation (droplet generation BSC).

6. Pre-warm the 96-well plate sealer by gently touching the screen.

Prepare the Serial Dilution

7. Place a 48-well dilution plate in a chilled 96-well freezer block and place in the dilution BSC.
8. Prepare 1X dilution buffer (see recipe above).
9. Pour the 1X dilution buffer into a polystyrene reagent reservoir.
10. Using a 20-200 µL multichannel pipette, carefully add the dilution buffer to the dilution plate according to the dilution scheme listed in step 12.
11. Use a single channel 1-10µl pipette to add 5 µL of each viral sample to Dilution 1 in the 48-well dilution plate and pipette 5-10 times to mix.
12. Dilute the virus as follows: Mix dilutions thoroughly, but pipette slowly to avoid generating aerosols.
• Dilution 1 (20X): 5 µL in 95 µL 1X PCR buffer (1:20)
• Dilution 2 (20X): 5 µL in 95 µL 1X PCR buffer (1:400)
• Dilution 3 (20X): 5 µL in 95 µL 1X PCR buffer (1:8,000)
• Dilution 4 (20X): 5 µL in 95 µL 1X PCR buffer (1:160,000)
• Dilution 5 (20X): 5 µL in 95 µL 1X PCR buffer (1:3,200,000)
• Dilution 6 (2X): 50 µL in 50 µL 1X PCR buffer (1:6,400,000)
• Dilution 7 (2X): 50 µL in 50 µL 1X PCR buffer (1:12,800,000)
• Dilution 8 (2X): 50 µL in 50 µL 1X PCR buffer (1:25,600,000)
13. Use multichannel pipettes for the dilution series.
• For dilutions 1-5, use the 1-10 µL multichannel pipette set to 5 µL.
• For dilutions 6-8, use the 20-200 µL multichannel pipette set to 50 µL.
• For mixing between dilutions, use the 20-200 µL multichannel pipette set to 90 µL and mix by pipetting the liquid up and down 10-20 times.
14. Gently cover the entire dilution plate with Microseal adhesive seal - do not press the film just gently cover so nothing falls into the plate.
15. Leave dilutions on the chilled 96-well freezer block in the BSC until ready to use.

Prepare the Master Mix

16. Place a ddPCR plate onto a chilled 96-well freezer block and set aside in the droplet generation BSC to cool.
17. Prepare the ITR master mix in a microcentrifuge tube as shown below. For 8 samples prepare enough master mix for 9 samples.

ITR Master Mix	Volume	9X Volume	Final Concentration
2X ddPCR Supermix for Probes, no dUTP	10 µL	90 µL	1X
10 µM ITR probe (FAM)	0.5 µL	4.5 µL	250 nM
Forward ITR Primer (10 µM)	1.8 µL	16.2 µL	900 nM
Reverse ITR Primer (10 µM)	1.8 µL	16.2 µL	900 nM
Nuclease-free water	5.9 µL	71.55 µL
Total Volume	20 µL

18. Vortex the master mix for 15 seconds and spin in a mini centrifuge for 10 seconds before use.
19. Place an 8-well PCR tube strip into a chilled 96-well freezer block.
20. Add 20 µL of the master mix to each PCR tube. Be careful to dispense to the bottom of the tube without collecting drops along the side of the tube.
21. Cap gently - do not push the cap in all the way, just ensure the samples are covered.
22. Bring the PCR tubes to the BSC used for dilutions.
23. Without disturbing the liquid in the tube, gently uncap the PCR tubes.
24. Add 5 µL of dilutions 6-8 to the appropriate PCR tubes. Pipette back and forth 5 times.
25. Lightly cap the PCR tubes.

Generate the Droplets

26. Bring the PCR tubes to the droplet generation BSC.
27. Without disturbing the liquid in the tube, gently uncap the tubes.
28. Add 5 µL nuclease-free water to the NTC tube.
29. Place a DG8 cartridge into the cartridge holder.
30. Using a 2-50 µL multichannel pipet, load 20 µL of the reaction mixtures into the middle wells of the cartridge.
31. Add 800 µL of droplet generation oil to a polystyrene reagent reservoir.
32. Using the 20-200 µL multichannel pipet, load 70 µL of droplet generation oil into the bottom row of wells.
33. Cover the cartridge with the DG8 gasket, making sure that it is secure.
34. Transfer the cartridge holder to the droplet generator. Close the lid and wait for the droplets to be generated.
35. Once the droplets have been generated, use a 20-200 µL multichannel pipet to aspirate 40 µL of droplets.

*Pro-Tip* To ensure that the droplets are not disrupted insert the pipette tips directly in the center of the well and tilt to a 45° angle. Count to 20 while slowly and gently aspirating the droplets.

36. Transfer the droplets to a prechilled PCR plate.

*Pro-Tip* To ensure that the droplets are not disrupted insert the pipette tips and gently touch the bottom of the well. Lift the tips ~1 mm. Touch the side of the well and tilt the pipette tips at a 45° angle. Count to 20 while slowly and gently dispensing the droplets down the side of the tube.

37. Place a Pierceable Foil Heat Seal on the PCR plate with the red line facing up. If the plate sealer is not at temperature, touch the screen on the plate sealer to allow it to get to temperature. Once the temperature is reached, place the PCR plate with the foil onto the metal support block. Place the block in the plate sealer and press the ‘Seal’ button.
38. After the plate has been sealed, proceed to thermocycling.

Thermal Cycling

39. Run the following PCR parameters.

Cycling Step	Temperature (°C)	Time (min)	Ramp Rate (°C/sec)	# Cycles
Denaturation	95	10	2	1
Denaturation	95	0.5	2	50
Annealing/Extension	60	1	2	50
Signal Stabilization	98	10	2	1
Hold	4	∞	2	1

40. After PCR is complete, transfer the plate to the Droplet Reader.
41. Open the QuantaSoft software to set up a new plate layout. Designate the sample name, experiment type, supermix type (ddPCR Supermix for Probes), the target names and target types.
42. When the plate layout is complete , select 'Run' to begin the droplet reading.
43. When the droplet reading is complete, export the data from all wells as a CSV file and use the following equation to calculate the titer for each sample:
$$T = {R*C*D*1000\over V}$$

Where:

• T = Titer, GC/mL
• R = Reaction volume (20 µL)
• C = Copies/µL
• D = Dilution factor of virus
• V = Volume of virus in reaction mix (4 µL)

Sample Data

• When analyzing data there should be a clear distinction between negative droplets (black) and positive droplets (blue).
• The no template control (NTC) should be close to zero (B08). At Addgene, we consider runs with an NTC > 5 to be invalid.
• To reduce NTC values, we recommend wiping down all pipettes and equipment with 10% bleach prior to use and keeping all reagents and samples on ice or pre-chilled 96-well freezer blocks during use.
• In this protocol, a dilution series is prepared for each AAV sample and the three final dilutions are assayed. The samples that are assayed are diluted 2-fold serially, therefore the concentration obtained by ddPCR should decrease by a factor of 2 across the dilutions.
• In the example below, 2-fold serial dilutions of a sample were loaded in wells A04, A05 and A06. As shown in the image (Figure 1) and table below, the concentration of positive droplets decreases by a factor of ~2.
• To increase the accuracy of the titer, take the average of several measurements.