Introduction
This protocol quantifies AAV genomic titer using ddPCR. The assay is optimized for BIO-RAD QX200 with an automated droplet generator.
Last Modified: May 13, 2024
Equipment
- BIO-RAD automated droplet generator
- BIO-RAD QX 200 reader
- BIO-RAD incubator
Reagents
- DNase I
- 10% Pluronic-F68
- Nuclease-free water
- Primers/probe targeting specific gene
- BIO-RAD ddPCR supermix
- 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)
- Internal AAV reference [AAVnerGene, SKU:DJ001001-AAV9]:
- Dilution buffer: Prepare 0.1% Pluronic-F68 in water
Digestion
- Treat AAV samples and AAV reference with DNase I: Add 90 μL DNase I solution to 10 μL AAV sample, 37℃ for 60 min. If the next step not be carried out soon, it would be better to store the digested sample at -80℃.
Dilution
Dilute the digested samples according to the dilution scheme in the table below.
| Est Titer, GC/mL | Up_5000 | Bottom_200 | D-1 | D-2 | D-3 | D-4 |
| 3.00E+14 | 1200k | 30000k | 1200k | 3600k | 10800k | 32400k |
| 1.00E+14 | 400k | 10000k | 400k | 1200k | 3600k | 10800k |
| 3.00E+13 | 120k | 3000k | 120k | 360k | 1080k | 3240k |
| 1.00E+13 | 40k | 1000k | 40k | 120k | 360k | 1080k |
| 3.00E+12 | 12k | 300k | 12k | 36k | 108k | 324k |
| 1.00E+12 | 4k | 100k | 4k | 12k | 36k | 108k |
Set up and load plate
- Prepare ddPCR reaction mix
- Based on the total number of reactions (Table 1 +10% excess, ensure all 8 wells within a single column receive 20 µL of supermix.), thoroughly premix the following components in a nuclease-free tube:
- ddPCR Supermix for Probes
- Forward/Reverse Primers
- Probe
- Nuclease-free water
- Based on the total number of reactions (Table 1 +10% excess, ensure all 8 wells within a single column receive 20 µL of supermix.), thoroughly premix the following components in a nuclease-free tube:
- Aliquot master mix
- Dispense 20 μL of the master mix into each well of a 96-well PCR plate
- Add template DNA
- Add 5 μL of diluted sample (or standard/control) per well (final volume: 25 μL)
- For NTC (No-Template Control), use 5 μL nuclease-free water
- Mix and centrifuge
- Mix gently: Pipette up/down 10 times (avoid bubbles)
- Seal plate with foil using PX1™ sealer (180°C, 5 sec)
- Spin down: Centrifuge the plate (1000 ×g, 30 sec) to settle all liquid
Droplet Generation
- Ensure the automated droplet generator (QX200™) is powered on and ready for operation
- Label a fresh 96-well PCR plate for droplet collection
- Immediately seal plate with foil using PX1™ sealer (180°C, 5 sec)
PCR Amplification (T1000 Cycler)
Run amplification using the cycling conditions in Table 2.
Note: Adjust annealing temperature based on primer/probe Tm.
Example of 96-well-plate set-up:
Droplet Reading (QX200 Reader)
- Load plate into QX200
- Open QuantaSoft™ Software, select:
- Experiment Type: Absolute Quantification (Probe).
- Detection Channels: FAM (Target) and HEX (Reference, if duplex).
- Run analysis (~2 min/well).
Data Analysis
- Thresholding: Auto-set or manually adjust to separate positive/negative droplets.
- Concentration: calculated by software (copies/μL).
- For AAV: GC/mL = (copies/μL) × digestion factor (10) × dilution factor × 1000.
- QC criteria:
- NTC: ≤10 positive droplets.
- ≥10,000 droplets/well.