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Innovative Medicines & Omics Economical method for titering viral vectors

(copies/mL) to generate a standard curve. The mean Ct 3. Results
value for each sample was then interpolated on the standard
curve to determine the copy number and titer (copies/mL). 3.1. Performance comparison of two lysis buffer
If the samples had been diluted before PCR, the final formulations for RNA preparations
concentration and/or titer were adjusted according to the The titers of vector stocks produced using either method
appropriate dilution factor. were not significantly different. The titer obtained from
the ABM kit was 32.6 ± 8.9 × 10 transduction units
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2.4. Statistical analysis (TU)/mL, whereas the titer from the TRIzol method
Data (n = 9 for each method) were subjected to statistical was 32.1 ± 4.6 × 10 TU/mL (n = 9). Table 3 shows the
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analysis to determine whether there was a significant concentration and purity of RNA isolated using the
difference in the viral titers obtained by the two methods. ABM kit, following the manufacturer’s instructions,
A Mann–Whitney U test was applied to the data to while Table 4 presents the same data for the five samples
identify significant differences. The standard curves processed using the TRIzol method. Statistically, the
were constructed using Graph Pad Prism7 software A260/A280 and A260/A230 ratios were significantly lower
(United States) and were used for interpolation and titer for the samples processed with the ABM kit compared to
calculations only if the R value was >0.99. those extracted using the TRIzol method (P < 0.0001).
2
The expected A260/230 ratios for “pure” nucleic acid are
Table 2. Real‑time PCR components for one well and qPCR typically in the range of 2.0 – 2.2. A ratio lower than this
amplification parameters. (A) PCR components for one well, may indicate the presence of contaminants. The higher
and (B) standard cycling conditions (primer temperature A260/A230 ratios obtained with the TRIzol method were
≥60°C) not significantly different from those of the ABM kit

(A) PCR components for one well method but were closer to the expected values for “pure”
PCR component Volume nucleic acid. These ratios did not impact the PCR process
Standards Samples in the titering assay. Moreover, the commercial lentiviral
preparations used in this study have been successfully
PowerUp SYBR Green 10 µL 10 µL used in in vitro (cell culture) and in vivo transduction
Master Mix (2×) experiments without issue.
Forward primer 0.25 µL 0.25 µL
Reverse primer 0.25 µL 0.25 µL 3.2. Comparison of lentiviral vector titration using
Template 5 µL 1 µL the TRIzol method and the commercial kit
Nuclease free water 9.5 µL 13.5 µL Figures 2 and 3 show the qPCR results for the ABM kit
Total 25 µL 25 µL and the TRIzol method, respectively, applied to nine
(B) Standard cycling conditions (primer temperature ≥60°C) samples for lentiviral titer determination. Following
Step Temperature Time Number of cycles the manufacturer’s instructions for the Lentivirus Titer
Reverse transcription 42°C 20 min 1 Kit (ABM; LV900; Canada), standards (supplied with
the kit) were diluted in the range of 10 , 10 , 10 , and
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Enzyme activation 95°C 10 min 1 10 copies/mL, followed by qPCR. Samples producing
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Denaturation 95°C 15 s 40 small pellets were diluted 1:1,000 times, whereas those
Annealing/extension 60°C 1 min producing large pellets were diluted 1:10,000. An R of
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Abbreviation: PCR: Polymerase chain reaction. 0.998 was obtained for the standard curve. For the TRIzol
Table 3. Summary of the concentration and purity of isolated viral ribonucleic acid using the quantitative polymerase chain
reaction lentivirus titer kit
Sample Lysis buffer (µL) Vector volume (µL) Vector RNA concentration (ng/µL) A260/280 ratio A260/230 ratio Total volume (µL)
1 18 2 30.8 0.39 0.08 20
2 18 2 48.2 0.53 0.11 20
3 18 2 30.1 0.38 0.08 20
4 18 2 43.3 0.51 0.11 20
5 18 2 29.5 0.36 0.08 20
Abbreviation: RNA: Ribonucleic acid.

Volume 2 Issue 1 (2025) 89 doi: 10.36922/imo.6552