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Quantitative PCR method for AAV Biodistribution Studies

Adeno-associated virus (AAV)-derived vectors are among the most promising gene therapy tools because of their safety and ability to mediate long-term transgene expression. The quantification of viral nucleic acid by quantitative polymerase chain reaction are widely employed to study the biodistribution of AAV products. The procedure by Betroff et al is described below as an example.

Collection and Preparation of Animal Tissues:

Homogenize the frozen animal organs by applying mechanical force using liquid nitrogen and a pestle to obtain a fine dry powder. 

Extraction of Genomic DNA extraction 
Perform the extraction of DNA using using the protocol of the DNeasy Blood & Tissue Kit (“Purification of Total DNA from Animal Tissues (Spin-Column Protocol)” in the DNeasy Blood & Tissue Handbook; QIAGEN, Hilden, Germany).
  • Add 20 µl Proteinase K to the tissue homogenate. Mix thoroughly by vortexing, and incubate at 56°C until the tissue is completely lysed. 
  • Vortex occasionally during incubation to disperse the sample or place in a thermomixer, shaking water bath or on a rocking platform.
  • Add 4 µl RNase A (100 mg/ml), mix by vortexing, and incubate for 2 min at room temperature (15–25°C) to degrade any RNA.
  • Vortex for 15 s. Add 200 µl Buffer AL to the sample, and mix thoroughly by vortexing. Then add 200 µl ethanol (96–100%), and mix again thoroughly by vortexing.
  • Place the DNeasy Mini spin column in a new 2 ml collection tube (provided), add 500 µl Buffer AW1, and centrifuge for 1 min at ≥6000 x g (8000 rpm). Discard flow-through and collection tube.
  • Place the DNeasy Mini spin column in a new 2 ml collection tube (provided), add 500 µl Buffer AW2, and centrifuge for 3 min at 20,000 x g (14,000 rpm) to dry the DNeasy membrane. Discard flow-through and collection tube. 
  • Place the DNeasy Mini spin column in a new 2 ml collection tube (provided), add 500 µl Buffer AW2, and centrifuge for 3 min at 20,000 x g (14,000 rpm) to dry the DNeasy membrane. Discard flow-through and collection tube.
  •  For maximum DNA yield, elution is performed in two successive steps using 200 µl Buffer AE each. For more concentrated DNA, elution can be performed in two successive steps of 100 µl each. Keep in mind that elution volume and number of elution steps depends on the amount of DNA bound to the DNeasy membrane
Quantification of Isolated Genomic DNA 
  • Measure the quantity of isolated genomic DNA with a NanoQuant Plate (Tecan, Männedorf, Switzerland) in combination with an Infinite 200 plate reader (Tecan, Männedorf, Switzerland).
  • The DNA concentration of samples was adjusted by dilution with TE buffer solution (pH 8.0) (Sigma-Aldrich, Munich, Germany) to the desired concentration of 50 ng/μL following storage at 2°C–8°C before qPCR analysis.
Design and Synthesis of primers and probe
The primers and probes are designed using the tools such as Primer Quest from IDT DNA.
The probes are labeled with carboxyfluorescein (FAM) at the 5′ end and the Eclipse quencher (Eurofins Genomics, Ebersberg, Germany) attached to the minor groove binder molecule at the 3′ end. (More details on Primer/Probe Design)

PCR Reaction 
The LightCycler 480 Probes Master Mix is a ready-to-use hot-start PCR mix and contains FastStart Taq DNA Polymerase, reaction buffer, deoxyribonucleotide triphosphate (dNTP) mix with deoxyuridine triphosphate (dUTP) instead of deoxythymidine triphosphate (dTTP), and 6.4 mM MgCl2.

The qPCR reaction mix (20 μL total volume/well) included 
  • LightCycler 480 Probes Master Mix  at 1× concentration, 
  • 400 nM hAlu fwd and rev primer, 50 nM probe,
  •  PCR-grade water (Roche Diagnostics, Mannheim, Germany), and 
  • 100 ng genomic DNA
qPCR Program
The qPCR program consisted of
  • 1 preincubation step of 50°C for 2 min; 
  • 1 initial denaturation step of 95°C for 5 min,
  • 45 cycles of 95°C for 10 s, 
  • 60°C for 30 s for annealing and amplification
  • 1 cycle of 40°C for 10 s for cooling. 

qPCR Data Analysis: 
The applied qPCR analysis program in the LightCycler 480 software, v.1.5.1.62, and monocolor hydrolysis detection format should be used. The Absolute Quantification/Second Derivative Maximum analysis method should be used for determining Cq and concentration values. The method takes the shape of the calibration curve into account when calculating the DNA concentration of a sample. Therefore, an error value is calculated for the calibration curve instead of the correlation coefficient. The correlation coefficient describes the correlation of data points with a monotonic decreasing or increasing function. 

Reference
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7327859/
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