We measured transduction efficiency by expression levels of d2Venus using FACS.
First, different cell lines can have different transduction efficiencies, therefore measuring transduction efficiencies in each cell line is critical, and can be done using either a GFP reporter or an empty vector and selecting for drug resistance.
One of the major differences in the approach using oncolytic RCAd11pGFP compared to traditional, replication-defective Ad5 vectors is that RCAd11pGFP can provide an opportunity to measure the transduction efficiency, and it can also replicate and produce progeny viruses.
The ratio of GFP-positive to Hoechst-positive objects was used as the measure for transduction efficiency and recorded as percentages.
To measure the photothermal transduction efficiency, the nanoparticle solution was maintained under magnetic stirring in a 1 cm path length quartz cuvette that was clamped to a foam cap to reduce the heat loss.
To measure adeno-associated virus transduction efficiency, sonicated adeno-associated virus samples were incubated with HT-1080 fibrosarcellscells (29).
Table 2 shows the transduction efficiency (measured as frequency of positive cells), its quantification technique, viral system used, MOI, and blood monocyte-derived macrophage differentiation method reported by each publication [ 12- 14, 19- 28, 28- 34].
Strikingly, miR-21 over-expressing Hela cells formed significantly less colonies during selection of stable clones, and colonies were also significantly smaller (Fig.S4B,C), even though transduction efficiency, measured as percentage of GFP-positive cells 48-h post-transduction, was higher for miR-21 than for miR-control (Fig. S4D).
