Skip to main content

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/

Labels

Labels:

Popular posts from this blog

Human Genome Editing: FDA Draft Guidance Summary

Consideration for Developing Gene Editing Product  1. Genome Editing Methods: Genome editing can be achieved through nuclease-dependent or nuclease-independent methods. Nuclease-dependent methods involve introducing site-specific breaks in DNA using technologies like zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), modified-homing endonucleases, and CRISPR-associated (Cas) nucleases. These breaks can lead to modification of the DNA sequence at the cleavage site. Nuclease-independent methods can change DNA sequences without cleaving the DNA and include techniques like base editing and synthetic triplex-forming peptide nucleic acids. The choice of GE technology should consider factors such as the mechanism of action, the ability to target specific DNA sequences, and the potential to optimize components for efficiency, specificity, or stability. 2. Type and Degree of Genomic Modification: Different GE approaches rely on DNA repair pathways such a...
Read more

Stem-Loop PCR for siRNA

 Stem-loop PCR is a method often used for detecting and quantifying small RNAs, such as siRNA or miRNA, which are typically difficult to amplify directly due to their short lengths. The method involves the design of a stem-loop reverse transcription (RT) primer, which enhances specificity and stability of the short RNA during the RT-PCR process, allowing for sensitive detection and quantification of the siRNA. Here’s a detailed guide to how stem-loop PCR can be applied to siRNA detection: Key Steps in Stem-Loop PCR for siRNA Designing the Stem-Loop RT Primer : Structure : The stem-loop RT primer consists of a loop region flanked by complementary sequences on either side (the "stem"), which will fold back on itself to form a hairpin structure. Specific Binding Region : A short sequence complementary to the 3’ end of the siRNA is added at the end of the stem-loop primer to ensure specific binding to the siRNA target. Stabilization : The loop structure helps prevent primer-dimer...
Read more

Stem loop RT-PCR for Detection of siRNA in Animal Tissues

Step Loop RT-PCR for Detection of Small Interfering RNA (siRNA) The recent publications described a novel used the novel method for the detection of siRNAs using a TaqMan®-based approach. This approach utilizes similar strategy that has been used for microRNA detection. The approach is illustrated in below.  In brief, the RT step occurs in the presence of a stem-loop RT primer that is complementary to the last 6–10 bases of the 3′ end of the antisense strand of the target siRNA. The stem-loop primer contains an additional universal sequence at the 5′ end that facilitates a TaqMan-based detection strategy in the subsequent qPCR step. As in the case of microRNA, the forward primer for qPCR is sequence-specific for the target siRNA. For sequence compositions that yield a low predicted melting temperature (Tm), the forward primer is designed as a tailed primer to help increase Tm. Stem Loop PCR for SiRNA Detection Step 1: Preparation of liver and plasma samples for the quanti...
Read more

Allometric scaling in AAV gene therapy dose estimation

 Allometric scaling in AAV gene therapy dose estimation is crucial for translating effective and safe doses from animal models to humans. Since AAV dosing often involves high viral vector concentrations, proper dose scaling is essential to minimize adverse effects and optimize therapeutic outcomes. Here’s a breakdown of how allometric scaling is applied in AAV gene therapy dosing and the considerations involved: 1. Concept of Allometric Scaling Allometric scaling is a method of adjusting drug doses across species based on body size, physiology, and metabolism. It is especially useful in biologics and gene therapies where the pharmacokinetics and pharmacodynamics are more complex than small-molecule drugs. For AAV vectors, dosing is commonly scaled by body weight (e.g., vector genomes [vg] per kilogram) or body surface area (BSA), as these parameters can approximate dose distribution and vector exposure across different species. 2. Standard Scaling Approaches Body Weight Scaling (mg...
Read more

Guideline on development and manufacture of lentiviral vectors (CHMP/BWP/2458/03)

The guideline with the reference number "CHMP/BWP/2458/03" pertains to the "Guideline on Development and Manufacture of Lentiviral Vectors." This guideline was developed by the Committee for Medicinal Products for Human Use (CHMP) and the Biotechnology Working Party (BWP) of the European Medicines Agency (EMA). It provides recommendations and regulatory guidance for the development and manufacture of lentiviral vectors, which are widely used in gene therapy and cell therapy applications. Here's an overview of the key points covered in this guideline: 1. Introduction: The guideline begins with an introduction highlighting the increasing importance of lentiviral vectors in advanced therapies and the need for guidance on their development and manufacture. 2. Scope: It defines the scope of the guideline, which covers the development and manufacture of lentiviral vectors intended for use in gene therapy and cell therapy products for human use. 3. Quality and Characte...
Read more