- AAV titration by qPCR with SYBR Green technology
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The AAV titration protocol can be used to determine the number of genomic particles in an AAV preparation using SYBR-Green technology.
This protocol was validated using an internal reference AAV of known titer, 100837-AAV1, and measuring the titer of samples obtained from academic viral vector cores. Our titers were similar to those reported by these institutions.
This protocol is for a 96-well plate with a 20 µL reaction volume.
- Last modified: February 13, 2019
- Estimated time required: ~3 hours
Watch the protocol video below to see how the antibiotic is spread onto agar.
- Plate configuration:2 hours
- qPCR execution:1.5 hours
- Data analysis:30 minutes
- Multichannel pipette (optional but strongly recommended)
- SYBR 2X Universal-Mastermix
- First pair targeting AAV2 ITR (Aurnhammer et al., 2012)
- front ITR choke, 5'-GGAACCCCTAGTGATGGAGTT
- rev ITR-Primer, 5'-CGGCCTCAGTGAGCGA
- Plasmid containing ITR for the standard curve
- RNase-free DNase
- 10X DNase-Puffer
- Nuclease-free water
- Microcentrifuge Tube
- 96-well optical plate
- pipette tips
- Always run standards and samples at least in duplicate
- If possible, include a reference sample of AAV with a known title. The reference material should have a titer within 1 log of the expected titer of the samples to be tested.
- Always add a no-template control (NTC), i.e. H. Master mix + water
- Whenever possible, use a multichannel pipette to minimize pipetting error and variability.
- Mix the samples very well by pipetting back and forth several times at each step.
Preparation of the reagents
Mastermix:Count the number of samples (n) and prepare the master mix for 10 additional samples (n+10: the additional number ensures that there is enough master mix for all samples). 15 µl master mix is required for each sample.
|Reagent||amount for ONE reaction||Amount for 100 reactions (1 96-well plate)|
|Universal-SYBR-Master-Mix 2X||10 l||1000 l|
|Forward-Primer 100 μM||0,15 l||15 l|
|100 μM inverse cooler||0,15 l||15 l|
|Nuclease-free water||4,7 l||470 l|
- Prepare a 2x10 plasmid broth9Molecules/μL to construct a standard curve:
- One option is to use plasmid#59462by Addgene. The values marked in red below were calculated with this plasmid, but will change if you use a different plasmid.
- To get a solution in 2x109Molecules/μl:
- 1,59 x 1011/ 2x109= 79.8-fold dilution 100 µL / 79.8 = 1.25 µL
- Therefore, we need to dilute 1.25L of stock into 98.74L of H20
sample calculations Addgene plasmid size in bp#59462: 6208 pb Addgene plasmid concentration#59462: 1.07 µg/µl molecular weight: 6208 pbx 650 Dalton/Pb (g/mol)=4,03x106grams/mole Mol/μl: 1.07 µg/µlx 1g/106µg x1 /4,03 x 106g/Mol=2,65x10-13Mol/μl Molecules/ μl: 2,65x10-13Mol/μlx 6,022145 x 1023molecules/mole =1,59 x 1011molecules/μl
- Once a validated standard curve is obtained, make a small aliquot of each standard (enough for 1-2 plates) and store at -20 °C. Once thawed, do not refreeze standards, store at 4°C and use within 1 week.
- Track the Ct value for each standard over time. They must remain within 0.5 cents of their initial value. If the standard's Ct deviates, it's time to make a new one.
- In developing the assay, several ITR-containing plasmids were tested. plasmid#59462it is a plasmid that gave reliable and consistent results. Use the recommended plasmid or try several plasmids to find a suitable one.
- Some labs have reported better results when the plasmid is linearized.
- Treat purified AAV samples with DNase I to remove contaminating plasmid DNA carried over from the production process (DNase does not enter the virion).
- 5 μL Probe + 39 μL H2ODER + 5 μl 10x DNase-Puffer + 1 μl DNase
- Mix sample gently (do not vortex)
- Incubate for 30 minutes at 37°C
- transferred to ice
** Critical: DO NOT treat your plasmid standard with DNase **
- Make 6 serial dilutions in duplicate of your standard curve plasmid (2x109stick from step #1):
Volume 2x109Stock or previous dilution (μL) Volume of nuclease-free water (uL) molecules per μl 10 90 2x108 10 the 2x108dilution 90 2x107 10 the 2x107dilution 90 2x106 10 the 2x106dilution 90 2x105 10 the 2x105dilution 90 2x104 10 the 2x104dilution 90 2x103
*Pro tip*To stabilize the standards, add carrier DNA to each standard dilution at a final concentration of 4 µg/mL.
- Dilute DNase and AAV treated reference samples according to the dilution scheme in the following table:
dilution series Sample volume (uL) Volume of nuclease-free water (uL) dilution factor full dilution Dilution 1 (DNase step) 5 ul Reserve of AAV 45ul 10x 10x dilution 2 Dil. 5ul 1 95ul 20x 200x dilution 3 20 µl kill. 2 80 st 5x 1000x dilution 4 20 µl kill. 3 80 st 5x 5000x dilution 5 20 µl kill. 4 80 st 5x 25000x dilution 6 20 µl kill. 5 80 st 5x 125000x dilution 7 20 µl kill. 6 80 st 5x 625000x dilution 8 20 µl kill. 7 80 st 5x 3125000x
The dilutions highlighted in green are those that will be loaded onto the qPCR plate for the majority of samples.
- If the sample is expected to have a titer <1x1012GC/mL, use 3-6 dilutions
- If the sample is expected to have a titer >3x1013GC/mL, use 5-8 dilutions
Note: In Addgene, the samples are usually between 1x1012GC/ml a >2x1013GC/mL and we use an internal reference virus which is 1x1013gc/mL. We always recommend using a reference within 1 log of the expected title.(Video) AAV Purification
- The quality of the sample dilution series is critical. Make sure to pipette each dilution up and down at least 10 times and use at least half of the final volume (mix with >50 µl if your well is 100 µl).
- Use a multichannel pipette to load the standards and samples onto the qPCR plate
- Set up and load the 96-well plate:
- Load 5 μL of each standard in duplicate.
- Load 5 µL of each sample in duplicate. Don't forget to use an untemplated control (NTC = Mastermix + Wasser).
- Add 15 µL of Master Mix per well and mix well by pipetting back and forth at least 5 times.
- Seal the plate with a transparent film.
- Centrifuge at 3000 rpm for 2 min to bring the sample to the bottom of the tube.
- Run the following protocol on your qPCR device with SYBR detection:
- 98 °C 3 min / 98 °C 15 s / 58 °C 30 s / read plate / repeat 39 times from step 3 / melting curve
Example panel mounting:
1 2 3 4 5 6 7 8 A 1.00x109 1.00x108 1.00x107 1.00x106 1.00x105 1.00x104 file NTC B 1.00x109 1.00x108 1.00x107 1.00x106 1.00x105 1.00x104 file NTC C AAV reference Probe 3 D mi Probe 1 Probe 4 F mi Probe 2 Probe 5 F
- Perform data analysis using the instrument software. Determine the physical titer of the samples (viral genomes (vg)/mL) based on the standard curve and the sample dilutions.
*Pro Tips*Make sure the qPCR is valid by checking the following:
- Standard curve: R2(correlation coefficient) ~1.0, E (PCR efficiency) ~100% (range from 90% to 110% is acceptable)
- Baseline Removal: All samples exhibit a small amount of background signal, which is most evident during the initial PCR cycles. This background signal must be removed to accurately determine differences between samples.
- Melting Curve Analysis: A single peak should be visible. The presence of a second peak at ~70-75°C usually indicates the presence of primer dimers, which can increase background signal and alter the Ct values of your samples.
- Quality of your standard curve: You should see differences in Ct values that make sense for your dilutions (a ~3.3 Ct difference for a 10-fold dilution is reasonable).
- Duplicate Quality: Exclude duplicates from analysis if they differ by more than 0.5 cents.
Illustration 1:Example of a valid 8-point standard curve.
Figure 2:Example of amplification plots obtained from an AAV sample. Each curve represents a dilution.
Aurnhammer C, Haase M, Muether N, Hausl M, Rauschhuber C, Huber I, Nitschko H, Busch U, Sing A, Ehrhardt A, Baiker A. Universal real-time PCR for the detection and quantification of adeno-associated virus serotype 2 - derived inverted terminal repeat sequences. Methods Hum Gene Ther. February 2012; 23(1):18-28.PMID: 22428977