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Innovative Medicines & Omics Economical method for titering viral vectors

A B

C

Figure 3. Quantitative polymerase chain reaction results for standards and lentiviral vector samples using the TRIzol method. (A) Amplification plots
for in-house standards (green), with concentrations from left to right: 10 , 10 , 10 , 10 , and 10 copies/mL. The curves in other colors represent viral
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samples. (B) Standard curve generated from the amplification plots of the prepared deoxyribonucleic acid standards. (C) Melt curves of amplified
products. R =0.998.
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kit used in this study provides a limited volume of 5. Conclusion
master mix (1.25 mL), primer mix (200 µL), standard
DNA (50 uL), and virus lysis buffer (800 uL). To We have developed a sensitive and accurate method
achieve accurate results, a viral range of 10 TU/mL is for measuring viral titers based on the use of WPRE as
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the template for SYBR Green-based real-time qPCR.
required, which often requires multiple dilutions of the This method provides a reliable assessment of lentiviral
samples (e.g., ×100, ×1,000, or ×10,000), consuming a copy number that is not significantly different from that
significant amount of the sample. In contrast, although
our technique also required sample dilutions to obtain obtained using a commonly used commercial kit. The use
the appropriate range, the use of TRIzol reagent is both of TRIzol to isolate lentiviral vector RNA for qPCR-based
more affordable and widely available (the procedure titration is a novel approach. Our method is comparable in
costs approximately AUD$115.00 for 96 samples). The time to the commercial kit but is significantly more cost-
effective, utilizing commonly available reagents found in
lysis buffer used to extract RNA was also significantly most molecular biology laboratories. This benefit makes it
better than that provided by the ABM kit. qPCR analysis an attractive alternative to more expensive commercial kits
using TRIzol yielded results that were accurate and not
significantly different from those obtained with the ABM for many research laboratories. Although the technique
kit. The significantly lower A260/A280 ratios observed with was initially designed for small research and clinical
the ABM kit may suggest the presence of contaminating laboratories, future studies could explore its scalability for
proteins, phenol, or other impurities in those samples. larger-scale processing, potentially incorporating robotics
or other forms of automation.
Similarly, the lower A260/A230 ratios in the ABM samples
may indicate contamination with guanidine hydrochloride Acknowledgments
or guanidine thiocyanate. While it was beyond the scope
of this study to investigate these contaminants further, None.
it would be of interest to test this methodology on other
retroviral and lentiviral vector systems in future studies. Funding
However, we anticipate that this would not present any This work was supported by the Ideas Grant (no. 1187040),
major issues, as the technique is simple and efficient for funded by the National Health and Medical Research
titering these vector systems. Council of Australia.

Volume 2 Issue 1 (2025) 91 doi: 10.36922/imo.6552