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Innovative Medicines & Omics Economical method for titering viral vectors

Table 4. Summary of the concentration and purity of isolated viral ribonucleic acid using the TRIzol method
Sample Vector volume (µL) Vector RNA concentration (ng/µL) A260/280 ratio A260/230 ratio Total volume (µL)
1 1 478.5 2.03 0.45 20
2 1 382.8 1.95 0.35 20
3 1 749.1 1.67 0.27 20
4 1 548.4 1.61 0.24 20
5 1 655.8 1.61 0.24 20
Abbreviation: RNA: Ribonucleic acid.

A B

C

Figure 2. Quantitative polymerase chain reaction results from the Applied Biological Materials kit. (A) Amplification plots for standards and lentiviral
vector samples. The amplification plots in green correspond to the standards provided in the kit (from left to right: 10 , 10 , 10 , 10 , and 10 copies/mL).
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The curves in other colors correspond to the viral samples. (B) Standard curve generated from the amplification plots of the provided deoxyribonucleic
acid standards. (C) Melt curves of amplified products. R =0.998.
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method (Figure 3), the qPCR standards were prepared quantification of integrated proviral DNA, titration
using plasmid DNA containing the WPRE element, along based on the expression of proteins linked to fluorescent
with the corresponding WPRE-specific primers (forward marker genes, and other methods using SYBR Green-
and reverse). qPCR was carried out using the Power Up based real-time qPCR with the WPRE template – similar
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SYBR Green Master Mix, and an R value of 0.998 was also to the approach used in this study – this paper highlights
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obtained for the standard curve. several unique aspects.
Both methods worked and produced similar In this work, we describe the development of a rapid,
results. The ABM kit calculated a titer of efficient, and economical method for titering third-
6.2 × 10 ± 2.3 × 10 copies/mL, whereas the TRIzol method generation lentiviral vectors. Although the method
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calculated 6.4 × 10 ± 2.3 × 10 copies/mL (n = 5). Analysis itself is not entirely novel, the use of TRIzol reagent in
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of the Ct values from the amplification curves indicated that this context is. Some traditional methods of lentiviral
the results from the two methods did not differ significantly. titration can take several days to complete, whereas
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commercial kits, such as the one tested here, are quick
4. Discussion but very expensive (AUD$1,958 for 96 samples).
While a large number of methods for lentiviral In addition, these kits provide reagents in specific,
titration has been described in the literature, including limited amounts and volumes. For example, the ABM
Volume 2 Issue 1 (2025) 90 doi: 10.36922/imo.6552