Addgene: AAV titration by qPCR (2023)

introduction

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

• qPCR-Instrument
• hotplate
• Pipetten
• Multichannel pipette (optional but strongly recommended)

General Considerations

• 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.

*Pro Tips*

• Use a SYBR “Universal” Master Mix containing a high quality DNA polymerase and dTTP/dUTP mix to minimize carryover contamination. The master mix should also include an internal passive reference (usually ROX dye) to normalize fluorescence fluctuations not attributable to PCR and to minimize well-to-well fluctuations resulting from a variety of causes such as: B. Pipetting errors and evaporation of the sample.
• Make the master mix after all samples have been added to the qPCR plate. Start by adding water, then SYBR Master Mix, then forward and reverse primer. Vortex briefly before use.
• Use a reservoir and a multichannel pipette to dispense the master mix into the wells.

Proceedings

1. 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.

   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

   • 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

   *Pro Tips*

   • 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.

2. Treat purified AAV samples with DNase I to remove contaminating plasmid DNA carried over from the production process (DNase does not enter the virion).
   1. 5 μL Probe + 39 μL H₂ODER + 5 μl 10x DNase-Puffer + 1 μl DNase
   2. Mix sample gently (do not vortex)
   3. Incubate for 30 minutes at 37°C
   4. transferred to ice

   ** Critical: DO NOT treat your plasmid standard with DNase **

3. 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.

4. 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

   *Pro Tips*

   • 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

5. Set up and load the 96-well plate:
   1. Load 5 μL of each standard in duplicate.
   2. Load 5 µL of each sample in duplicate. Don't forget to use an untemplated control (NTC = Mastermix + Wasser).
   3. Add 15 µL of Master Mix per well and mix well by pipetting back and forth at least 5 times.
   4. Seal the plate with a transparent film.
   5. Centrifuge at 3000 rpm for 2 min to bring the sample to the bottom of the tube.
   6. 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

6. 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.

sample date

references

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