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Innovative Medicines & Omics Economical method for titering viral vectors

Figure 1. Graphical representation of the TRIzol method of lentiviral vector titration. Created in BioRender by Ann M. Simpson (2024). https://BioRender.
com/t86k558.
ii. 1 µL of the diluted vector was added to 0.5 mL of xiii. The RNA pellet was resuspended in 20 µL of ultrapure
TRIzol reagent. The mixture was shaken vigorously DNase/RNase-free water
for 30 s to ensure complete lysis of the cells. The xiv. 1 µL was used to test the RNA quantity and quality
TRIzol solution, used in place of the ABM lysis through Nanodrop spectroscopy or another
solution, was superior for the lysis and extraction of appropriate method
viral RNA xv. The RNA was stored at −20°C or −80°C.
iii. The sample was incubated at room temperature for The vectors titered in this study included clinical-grade
8 min, with intermittent mixing, to allow complete samples that had been produced commercially for use in
dissociation of the nucleoprotein complex animal models at the Viral Vector Manufacturing Facility,
iv. 0.2 mL chloroform was added to the tube, mixed Westmead Health, New South Wales, Australia.
thoroughly by inverting several times, and incubated
at room temperature for 5 min 2.3.3. qPCR analysis
v. The sample was centrifuged at 21,100 × g for 15 min i. Using the WPRE-containing plasmid pRRLSIN.cPPT.
at 4°C PGKGFP.WPRE of known concentration and serial
vi. The colorless upper aqueous phase was transferred dilutions of the plasmid were prepared in the range of
into a new 2 mL microcentrifuge tube 10 – 10 copies/mL
5
9
vii. The RNA was precipitated by adding 0.5 mL of ii. Standards were amplified using the WPRE forward
isopropanol, mixing well by inversion, and the tube and WPRE reverses primers to generate a standard
was incubated at room temperature for 10 min curve
viii. The sample was centrifuged at 21,100 × g for 15 min iii. qPCR reactions were carried out using the Power Up
at 4°C SYBR Green Master Mix (2×) after reverse transcription,
ix. If the pellet was small and not visible, the aqueous following the manufacturer’s instructions
phase was carefully removed, leaving approximately iv. Reactions were prepared by pipetting from a master
200 µL of liquid in the tube without disturbing the mix, which was made for the appropriate number of
pellet reactions to be analyzed (standards and viral vector
x. 1 mL of 75% (volume/volume) ethanol was added to samples). Table 2 lists the components of one well.
the tube, and the mixture was inverted 3 – 5 times
xi. The sample was centrifuged at 211,309 × g for 10 min 2.3.4. Calculating lentiviral titer
at 4°C All standards and samples were analyzed by qPCR in
xii. The liquid was carefully aspirated and discarded, either duplicate or triplicate reactions. After qPCR, the
and the sample was allowed to dry for 5 min at room mean cycle threshold (Ct) values for the standards were
temperature plotted against the deoxyribonucleic acid (DNA) copies

Volume 2 Issue 1 (2025) 88 doi: 10.36922/imo.6552