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viral-icon-01.png AAV Titration by qPCR Using SYBR Green Technology


Introduction

This protocol can be used to determine the number of genome-containing particles in an AAV preparation (physical titer) using SYBR green technology. This protocol is for a 96-well plate with 20 μL reaction volume.

Last Upload: July 13, 2016

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Workflow Timeline

Plate set-up: 2 hours

qPCR run: 1.5 hours

Data analysis: 1 hour

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Equipment

  • qPCR instrument
  • Heating plate
  • Pipettors
  • Multi-channel pipette

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Reagents

  • Universal SYBR Master Mix 2X
  • Primer pair targeting AAV2 ITR
  • ITR-containing plasmid for standard curve
  • RNase-free DNAse
  • 10X DNAse buffer
  • Nuclease-free water
  • Microcentrifuge tubes
  • 96-well optical plate
  • Pipettes
  • Pipette tips

Reagent Preparation

Master Mix: Count the number of samples (n) and prepare master mix for an additional 10 samples (n+10, the additional samples will ensure that you don’t run out of master mix before the last sample due to pipetting error). You will need 15 μL of master mix per sample.

Reagent Amount for ONE Reaction Amount for 100 reactions (1 x 96 well plate)
Unversal SYBR Master Mix 2X 10 μL 1,000 μL
100 μM Forward Primer 0.1 μL 10 μL
100 μM Reverse Primer 0.1 μL 10 μL
Nuclease Free Water 4.8 μL 480 μL
Total Volume 15 μL 1,500 μL

Considerations Before You Start

  • Always run your standard and samples in at least duplicate.
  • Always include a No Template Control (NTC), i.e., master mix + water.
  • Whenever possible, use a multichannel pipette to minimize pipetting error and variability.
  • Mix your samples well by pipetting back and forth multiple times at each step.

Procedure

  1. Prepare a plasmid stock of 2x109 molecules /μl to generate a standard curve:

    • Note: This is an example using Addgene plasmid #51084. Values will change for your plasmid. In these calculations, we assume the average molecular weight for a base pair of DNA is 650 Da.

      Sample Calculations
      Plasmid Size:
      6,750 bp

      Plasmid Concentration:
      1.07 μg/μL

      Plasmid Molecular Weight:
      6,750 bp x 650 Da/bp (g/mol) = 4.39 x 1x106 g/mol

      Plasmid Moles/ μL:
      1.07 μg/μL x 1 g/1x106 μg x 1 / 4.39 x 1x106 g/mol = 2.44 x 1x10-13 moles/μL

      Plasmid Molecules/μL:
      2.44 x 1x10-13 moles/ μL x 6.022145 x 1x1023 molecules/mole = 1.47 x 1x1011 molecules/μL

  • To dilute this plasmid stock and obtain a 100 μL solution with 2x109 molecules/μL:

    (1.45 x 1x1011 plasmid molecules/ μL) / (2x109 plasmid molecules/ μL [desired concentration]) = 73.4x dilution

  • 100 μL / 73.4 = 1.36 μL

  • Therefore, we need to dilute 1.36 μL of stock plasmid into 98.64 μL H2O

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    *Pro-Tip* Re-measure the concentration (μg/μL) of the purified plasmid stock occasionally and re-make the 2x109 stock as needed. Concentration will change over time due to evaporation.
  • 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).

    • 5 μL sample + 12 μL H2O + 2 μL 10X DNase buffer + 1 μL DNAse
    • Gently mix sample (do not vortex).
    • Incubate 30 min at 37 ℃.
    • Transfer to ice.
  • **Critical: Do NOT treat your plasmid standard with DNAse**

  • Make 5 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 H2O (μL) Molecules/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 2x105
  • Dilute DNAse-treated samples:

    • 1:20 dilution: 5 μL sample + 95 uL of H2O
    • 1:100 dilution: 20 μL of 1:20 + 80 μL H2O
    • 1:500 dilution: 20 μL of 1:100 + 80 μL H2O
    • 1:2,500 dilution: 20 μL of 1:500 + 80 μL H2O
  • Set up and load the 96-well plate:

    • Load 5 μL of each sample in duplicate. Do not forget to include a no template control (NTC = master mix + water).
    • Add 15 μL of Master Mix and mix well by pipetting back and forth at least 5 times. Seal plate with transparent film.
    • Centrifuge at 2,415 g for 2 min to get the samples to the bottom of their wells.
  • Run the following protocol in your qPCR instrument using SYBR detection:

    98 ℃ 3 min / 98 ℃ 15 sec / 60 ℃ 30 sec / read plate / repeat 39x from step 3 / melt curve

  • Perform data analysis using your instrument’s software. Determine the physical titer of your sample (viral genomes (vg)/mL) based on your standard curve
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    *Pro-Tip*
    • Standard curve: Should have an R2 ~ 1.0 and an E (efficiency of PCR) of ~ 100%
    • 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 (a 10-fold dilution corresponds to a ~3.3 difference in Ct value).
    • Quality of duplicates: Exclude duplicates from analyses if there is more than a 0.5 Ct difference between them.
  • Sample Data

    qPCR of AAV Vector

    Figure 1: Sample amplification plots obtained from standard dilutions of an AAV sample. Each curve represents a single dilution. Ct values determined from similar plots were used to generate the standard curve shown in figure 2.

    Standard Curve for AAV Titration by qPCR

    Figure 2: Sample 8-point standard curve generated from qPCR of dilutions of plasmid #37825. Sample statistics are shown below the chart.