Figure 2.

Lenti/βAS3-FB Had Reduced Levels of Complete RNA in Packaging Cells and Vector Particles

(A) Schematic representation of viral RNA and the PCR primers and probes used to quantify each RNA species by ddPCR. Transcription starts at the 5′ LTR driven by the CMV promoter, and RNA is transcribed in the following order: R/U5, PBS, and U3/R. R/U5 primers were used to quantify initial RNA, PBS primers quantify intermediate RNA, and U/3R primers quantify complete RNA.

(B and C) (B) The absolute quantification of viral RNA in 293T packaging cells measured by ddPCR and (C) the percentages of intermediate and complete viral RNA in 293T cells. Cells were harvested 3 days after transfection. Total RNA was extracted from 293T cells, treated with DNase, and reverse transcribed with random primers. The amounts of viral RNA species were quantified by ddPCR. The percentage of intermediate RNA was calculated as intermediate RNA/initial RNA × 100%, and the percentage of complete RNA was calculated as complete RNA/initial RNA × 100%.

(D) The absolute quantification of viral RNA in unconcentrated viral supernatant measured by ddPCR.

(E) The percentages of intermediate and complete RNA in vector particles (B–E, bars represent mean with SEM; n = 5 dishes of identical cultures treated and analyzed in two independent experiments).