AAV Titration by qPCR Using SYBR Green Technology

Introduction

The AAV Titration protocol can be used to determine the number of genome-containing particles of an AAV prep using SYBR green technology.

This protocol was validated using an internal reference AAV of known titer, 100837-AAV1, and by 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 20 μL reaction volume.

• Last Modified: February 13, 2019
• Estimate time required: ~3 hours

Protocol Video

Watch this instructional video for advice on how to successfully set up your qPCR to determine the genome copies in a given AAV preparation.

• Plate set-up: 2 hours
• qPCR run: 1.5 hours
• Data analysis: 30 minutes

Equipment

• qPCR instrument
• Heating plate
• Pipettors
• Multi-channel pipette (optional, but strongly recommended)

Reagents

General Considerations

• Always run standards and samples in duplicate at least
• If possible, include an AAV reference sample of known titer. The reference material should have a titer within 1-log of the expected titer of the samples being tested.
• Always include a No Template Control (NTC), i.e master mix + water
• Whenever possible use a multichannel pipet to minimize pipetting error and variability
• Mix samples very well by pipetting back and forth multiple times at each step

Reagent Preparation

Master Mix: count the number of samples (n) and prepare master mix for an additional 10 samples (n+10 - the additional amount will ensure that there is enough master mix for all samples). Each sample requires 15 μL of master mix.

Procedure

1. Prepare a plasmid stock of 2x10⁹ molecules/μl to generate a standard curve:
   • One option is to use plasmid #59462 from Addgene. The values highlighted below in red were calculated using this plasmid, but will change if you use a different plasmid.

   Sample Calculations
   Size in bp of Addgene plasmid #59462:	6208 bp
   Concentration of Addgene plasmid #59462:	1.07 μg/μL
   Molecular Weight:	6208 bp x 650 daltons/bp (g/mole)= 4.03 x 106 grams/mole
   Moles/μl:	1.07 μg/μL x 1g/106 μg x1 / 4.03x106 g/mole = 2.65 x10-13 moles/ μL
   Molecules/ μl:	2.65 x 10-13 moles/ μL x 6.022145 x 1023 molecules/mole = 1.59x 1011 molecules/μL

   • To obtain a solution at 2x10⁹ molecules/μL:
     • 1.59 x 10¹¹ / 2x10⁹ = 79.8x dilution 100 μL / 79.8 = 1.25 μL
     • Therefore we need to dilute 1.25 μL stock into 98.74 μL H₂0
   *Pro-Tips*

   • Once a validated standard curve is obtained, make a small aliquot of each standard (enough for 1 or 2 plates) and store at -20°C. Once a standard is thawed do not freeze it again but store at 4°C and use within 1 week.
   • Keep track of the Ct value for each standard over time. They should remain within 0.5 Ct of their initial value. If the Ct value of the standard starts to drift, it’s time to make a new one.
   • When developing the assay multiple plasmids containing ITR were tested. Plasmid #59462 is one plasmid that gave reliable and consistent results. Use the recommended plasmid, or test multiple plasmids to find a suitable one.
   • Some labs have reported better results when the plasmid is linearized.
2. Treat the purified AAV samples with DNase I to eliminate any contaminating plasmid DNA carried over from the production process (DNase does not penetrate the virion).
   1. 5μL sample + 39μL H₂O + 5μL 10x DNase buffer + 1μL DNase
   2. Gently mix sample (do not vortex)
   3. Incubate 30 minutes at 37°C
   4. Transfer to ice

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

3. Make 6 serial dilutions, in duplicate, of your standard curve plasmid (2x10⁹ stock made in step #1):

   Volume of 2x109 stock or previous dilution (μL)	Volume of nuclease-free water (uL)	Molecules per μL
   10	90	2x108
   10 of 2x108 dilution	90	2x107
   10 of 2x107 dilution	90	2x106
   10 of 2x106 dilution	90	2x105
   10 of 2x105 dilution	90	2x104
   10 of 2x104 dilution	90	2x103

   *Pro-Tip* To help stabilize the standards add carrier DNA to a final concentration of 4 ug/mL to each standard dilution.

4. Dilute DNase-treated and AAV reference samples according to the dilution scheme in the table below:

   Dilution Series	Volume of sample(uL)	Volume of nuclease free water (uL)	Dilution factor	Total dilution
   Dilution 1 (DNase step)	5uL AAV stock	45 uL	10x	10x
   Dilution 2	5uL Dil. 1	95 uL	20x	200x
   Dilution 3	20uL Dil. 2	80 uL	5x	1000x
   Dilution 4	20uL Dil. 3	80 uL	5x	5000x
   Dilution 5	20uL Dil. 4	80 uL	5x	25000x
   Dilution 6	20uL Dil. 5	80 uL	5x	125000x
   Dilution 7	20uL Dil. 6	80 uL	5x	625000x
   Dilution 8	20uL Dil. 7	80 uL	5x	3125000x

   Dilutions highlighted in green are the ones loaded onto the qPCR plate for most samples.

   • If sample is expected to have a titer <1x10¹² GC/mL, use dilutions 3-6
   • If sample is expected to have a titer >3x10¹³ GC/mL, use dilutions 5-8

   Note: at Addgene, samples typically range from 1x10¹² GC/mL to >2x10¹³ GC/mL and we use an internal reference virus that is 1x10¹³ GC/mL. We recommend always using a reference within 1-log of the expected titer.

   *Pro-Tips*

   • The quality of the sample dilution series is critical. Make sure to pipet each dilution up and down at least 10 times, and use at least half of the final volume (mix with >50uL if your well contains 100uL)
   • 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. Do not forget to include a no template control (NTC = master mix + water).
   3. Add 15 μL of Master Mix per well and mix well by pipetting back and forth at least 5 times.
   4. Seal plate with transparent film.
   5. Centrifuge at 3,000 rpm for 2 min to bring the sample to the bottom of the tube.
   6. Run the following protocol in your qPCR instrument using SYBR detection:
      • 98°C 3 min / 98°C 15 sec / 58°C 30 sec / read plate/ repeat 39x from step 3 / melt curve

   Example of plate set-up:

   	1	2	3	4	5	6	7	8
   A	1.00 x 109	1.00 x 108	1.00 x 107	1.00 x 106	1.00 x 105	1.00 x 104	empty	NTC
   B	1.00 x 109	1.00 x 108	1.00 x 107	1.00 x 106	1.00 x 105	1.00 x 104	empty	NTC
   C	AAV reference				Sample 3
   D
   E	Sample 1				Sample 4
   F
   E	Sample 2				Sample 5
   F

6. Perform data analysis using the instrument’s software. Determine the physical titer of samples (viral genomes (vg)/mL) based on the standard curve and the sample dilutions.
   *Pro-Tips* Make sure that the qPCR is valid by checking to the following:

   • Standard curve: R²(coefficient of correlation) ~ 1.0, E (efficiency of PCR) ~100% (90%-110% range is acceptable)
   • Baseline removal: all samples will have some small amount of background signal that is most evident during initial PCR cycles. This background signal must be removed to accurately determine differences between samples.
   • Melt curve analysis: a single peak should be seen. The presence of a second peak at a temperature of ~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 observe differences in Ct values that make sense for your dilutions (~3.3 difference Ct for a 10-fold dilution is appropriate).
   • Quality of duplicates: Exclude duplicates from analyses if there is more than a 0.5 Ct difference between them.

Sample Data

Figure 1: Example of a valid 8-point standard curve.

Figure 2: Example of the amplification plots obtained from an AAV sample. Each curve represent a dilution.

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. Hum Gene Ther Methods. 2012 Feb;23(1):18-28. PMID: 22428977