TITLE: Absolute quantification of AAV genomes from total DNA with digital droplet PCR (ddPCR)
AUTHORS: Gerard Michael Coughlin

[DESCRIPTION]
Accurate and absolute determination of AAV transduction from total DNA is important for understanding gene transfer efficiency across multiple experimental manipulations. As compared to relative measurements (e.g. through comparison of qPCR CT values between experimental conditions), absolute quantification can provide biological insights (e.g. actual average MOIs) that can guide further development of these vectors. This protocol enables absolute measurement of AAV genome number from total DNA samples extracted from animal tissues. Because total DNA is extracted, it is possible to estimate the number of AAV genomes per cell, using DNA content per cell and an estimate of transduction efficiency. 

[STEPS]
SECTION: Reagent set-up
1. Before beginning, ensure that you are able to specifically detect your AAV genome and design PCR reactions. We recommend using probe-based assays and designing 2 separate assays against distinct sequences in your target genome, with 2 separate fluorophores to detect each target sequence separately. During analysis, only count spots positive for both target sequences as true positives.

 

SECTION: Sample preparation and total DNA extraction
6. Process samples with Qiagen DNeasy Blood and Tissue total DNA purification kit. Follow manufacturer instructions for total DNA extraction.

Measure [DNA] using a spectrophotometer. 

If processing immediately, total DNA samples can be kept on ice or at  4 °C until ready to proceed. Alternatively, store at -20 °C or -70 °C until ready to proceed. 

SECTION: Sample preparation and total DNA extraction
5. Begin tissue collection.  Euthanize animals according to your lab's approved protocol, then transcardially perfuse animals with 30 mL  of heparinized PBS. Working quickly, dissect out and place tissue of interest into a petri dish with ice-cold PBS, then isolate regions of interest. Dissect tissues of interest into 2-3 mm cubes (maximum 25 mg of wet tissue weight), and transfer to 1.5 mL tubes. 

If processing immediately, keep tissue on ice until ready to process (Step 6).

If not processing immediately, snap-freeze tissue by immersing tubes in the dry ice-ethanol slurry, and store on dry ice until ready to transfer to -70 °C . Once ready to begin processing, remove from -70 °C and proceed to step 6.

If also collecting tissue to fix, gently drop tissue into 4% PFA in 1x PBS, and post-fix at 4 °C   , then proceed with standard procedures. Fresh tissue may also be harvested and homogenized, dissociated or snap-frozen for other downstream analyses.
SECTION: Sample preparation and total DNA extraction
4. On day of harvest, prepare work area. You will need: 

ice-cold 1x PBS
ice-cold 1x heparinized PBS
clean 10 cm petri dishes
ice 
tools for perfusion and microdissection
1.5 mL tubes for tissue collection
dry ice (optional; if choosing to snap freeze tissue, rather than processing immediately)
dry ice-ethanol slurry (optional; if choosing to snap freeze tissue, rather than processing immediately)

Pre-label collection tubes. If planning to freeze tissue, use an ethanol-resistant marker

If analysis of fixed tissue is also desired (e.g. for detection of an AAV-encoded fluorescent protein), prepare tubes with appropriate volumes of 4% PFA in 1x PBS and keep on ice. In addition, tissue may be collected and homogenized, dissociated, or snap-frozen for other downstream analyses.

 

SECTION: Sample preparation and total DNA extraction
3. Transduce animals with AAVs, carrying sequence that will be amplified in droplet PCR reaction.
SECTION: Sample preparation and total DNA extraction
2. General note on sample preparation

 

SECTION: Restriction enzyme digest
7. Note about restriction enzyme digest

 

SECTION: Restriction enzyme digest
8. Choose restriction enzymes that cut AAV genome in or near ITR regions, and that do not cut within target amplicons. Ideally, choose enzymes that:

can be used in a single reaction
can be safely used in   digests
can be heat inactivated 

 

SECTION: Restriction enzyme digest
9. Set up digest reactions. 

Combine: 
Total DNA: 1000 ng 
Restriction enzyme 1: 20 U
Restriction enzyme 2 (if necessary): 20 U
Appropriate buffer (e.g. CutSmart): 2 µL 
UltraPure water: Bring to 20 µL 

Incubate in a thermocycler with a heated lid. Use manufacturer-suggested incubation temperature (e.g. 37 °C ), and heat-inactivate, if possible. 

To ensure complete digestion, we recommend digesting samples   (if allowable by chosen restriction enzymes). 

 

SECTION: Restriction enzyme digest
10. Following digest and heat-inactivation, samples can be stored at -20 °C or -70 °C , or at 4 °C for immediate processing. 
SECTION: DNA dilution and digital droplet PCR
11. Note about optimal dilution

 

SECTION: DNA dilution and digital droplet PCR
12. Make a plan for digital droplet PCR. We recommend running at least 2 technical replicates per sample, and multiplexing 2 separate target sequences in the same wells, using different fluorophores. Prepare extra to account for pipetting error. 

For each reaction, you will need:

Diluted DNA sample(s): 5 µL 
50x probe/primer mix 1: 0.5 µL 
50x probe/primer mix 2: 0.5 µL 
UltraPure water: 6.5 µL 
ddPCR Supermix for Probes: 12.5 µL 

Combine all components, except DNA samples. Aliquot 5 µL of DNA to PCR strip tubes, then add 20 µL of above mastermix. Mix by tapping and spin down. 

 

SECTION: DNA dilution and digital droplet PCR
13. Prepare droplets with 23 µL of PCR reaction and 70 µL of droplet generation oil. Follow manufacturer directions for droplet generation. 

Once droplets are generated, transfer 46 µL of droplets to PCR plate. Once all samples are prepared, seal PCR plate with a pierceable heat seal and run PCR according to manufacturer instructions. 

SECTION: DNA dilution and digital droplet PCR
14. Once PCR is complete, analyze droplets with droplet reader. Follow manufacturer directions for droplet reader.

Data can be analyzed using Bio-Rad QuantaSoft Software. Count droplets that are positive for both target sequences as true positives. 

The following example may help in converting from copies/µL in the ddPCR reaction to a more meaningful metric (copies/ng of original total DNA sample):

You digested 1000 ng  of total DNA in a 20 µL , then diluted that 1:10 and loaded 5 µL into a 25 µL ddPCR reaction. 23 µL of the ddPCR reaction was used to generate droplets, and 46 µL of emulsification was read by the droplet reader. 
After analysis, you measured 500 copies/µL . Note that this number corresponds to the concentration in the ddPCR mix.
If there were 500 copies/µL in the 25 µL ddPCR reaction, then there were 12500 copies total in the PCR reaction (500 x 25 = 12,500)
Those 12500 copies were loaded in 5 µL of diluted DNA sample; thus the diluted DNA sample had 2500 copies/µL (12,500 / 5 = 2500).
That diluted sample was a 1:10 dilution of the original, so the original sample had 25000 copies/µL
1000 ng of total DNA was digested in  a20 µL reaction, so the undiluted sample had a DNA concentration of 50 ng/µL .
Thus, the original sample had an AAV genome concentration of 25000 copies/µL / 50 ng/µL  = 500 copies/ng