Adenoviral siRNA expression reduces target mRNA levels in a time- and m.o.i.-dependent manner. (A) Northern blot analysis of adenoviral expression of siRNAs against GNAS (Ad-siRNA-GNAS) in U2OS cells (m.o.i. 3000) in a time course from day 1 to 10 postinfection (p.i.). The viruses were removed from the cells at day 3, and medium was refreshed at days 3, 6, and 8. Cells reached confluency at day 4. From day 8, cultures still appeared viable, although some cells detached. In spite of these observations, the RNA yield as well as the absolute Ct values of the GAPD mRNA did not show large variations between these samples, indicating no significant adverse effects during this long experiment. As control for equal loading of the gel, the results of rehybridization with a probe against endogenous U6 snRNA is given at the bottom. (B) Real-time RT-PCR expression analyses for GNAS mRNA of the same samples as used in panel A. Data were plotted relative to samples derived from uninfected cells. The results of the infections of Ad-siRNA-GNAS (solid bars) as well as for control virus (open bars) are given. As control virus, Ad-siRNA-GL2 is used for the data point of day 3, and Ad-siRNA-empty (a construct lacking a hairpin sequence downstream of the U6 promoter) for the other data points. The GNAS mRNA expression of each individual sample was normalized for GAPD. (C) Real-time PCR expression analysis for GNAS mRNA of samples derived from U2OS cells infected with Ad-siRNA-GNAS at m.o.i. 300, 1000, or 3000 harvested at 1, 2, 3, or 6 d postinfection. The values are plotted relative to samples derived from cells infected with control virus Ad-siRNAGL2 harvested at the same time points. Shown are representative results from independent experiments.
