TITLE: AAV Titration by qPCR Using SYBR Green Technology
AUTHORS: Addgene the nonprofit plasmid repository

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol goes through AAV titration by qPCR using SYBR Green Technology. To see the full abstract and additional resources, visit the </span><a href="https://www.addgene.org/protocols/aav-titration-qpcr-using-sybr-green-technology/" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Addgene protocol page</span></a><span style = ":;">.</span></div><div class = "text-block"><span style = "font-weight:bold;">Sample Data</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/public/5acd19e056bdf88808866a63dd76bebaf3bfacef4acb2f29cfb4ece3805005bf/bqis7esn.png" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/public/5acd19e056bdf88808866a63dd76bebaf3bfacef4acb2f29cfb4ece3805005bf/bqit7esn.png" /></div><div class = "text-block"><span style = "font-weight:bold;">References</span></div><div class = "text-block"><span>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.</span><a href="https://www.ncbi.nlm.nih.gov/pubmed/22428977" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">PMID: 22428977</span></a></div></div>

[STEPS]
?. [Procedure]
Prepare a plasmid stock of 2x109molecules/μl to generate a standard curve:To obtain a solution at 2x109 molecules/μL:1.59 x 1011 / 2x109 = 79.8x dilution 100 μL / 79.8 = Therefore we need to dilute  stock into  H20
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.
1.25 µl
1.25 µl
98.74 µl
*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  . Once a standard is thawed do not freeze it again but store at   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.
?. [Procedure]
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). sample +  H2O +  10x DNase buffer +  DNaseGently mix sample (do not vortex)Incubate   at  .Transfer to iceIncubate  at .** Critical: do NOT treat your plasmid standard with DNase **
5 µl
39 µl
5 µl
1 µl
37 °C
?. [Procedure]
Make 6 serial dilutions, in duplicate, of your standard curve plasmid (2x109stock made in step #1):
*Pro-Tip*To help stabilize the standards add carrier DNA to a final concentration of 4 ug/mL to each standard dilution.
?. [Procedure]
Dilute DNase-treated and AAV reference samples according to the dilution scheme in the table below:Dilutions highlighted in green are the ones loaded onto the qPCR plate for most samples.
If sample is expected to have a titer 12 GC/mL, use dilutions 3-6If sample is expected to have a titer >3x1013 GC/mL, use dilutions 5-8
Note, at Addgene we use 2 different AAV reference depending on whether we expect our sample(s) to have a low (12) or high titer (>7x1012).Low titer reference: AAV RSM2, 3.28 x 1010 GC/mL, available from ATCC (cat# VR-1616)High titer reference: internal AAV vector, 3.0 x1013 GC/mL
*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 >  if your well contains  )Use a multichannel pipette to load the standards and samples onto the qPCR plate
?. [Procedure]
Set up and load the 96-well plate:Load  of each standard in duplicateLoad  of each sample in duplicate. Do not forget to include a no template control (NTC = master mix + water).Add  of Master Mix per well and mix well by pipetting back and forth at least 5 times.Seal plate with transparent film.Centrifuge at 3,000 rpm for   to bring the sample to the bottom of the tube.Run the following protocol in your qPCR instrument using SYBR detection:Centrifuge at 3,000 rpm for  to bring the sample to the bottom of the tube. for  /  for  /  for  / read plate/ repeat 39x from step 3 / melt curveExample of plate set-up:
5 µl
5 µl
15 µl
98 °C
98 °C
58 °C
?. [Procedure]
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.