Figure 6.

The effect of siRNA knockdown in conforming the specificity of MB1 (B, C) and MB8 (D, E). HDF cells with upregulated BMP-4 level were transfected with either nonsilencing siRNA or BMP-4 siRNA 4 h before adenovirus infection. (A) The effect of siRNA in knocking down BMP-4 mRNA level as measured by RT–PCR. The bar graph shows an ∼90% knockdown in the BMP-4 mRNA level as compared with that of control cells transfected with nonsilencing siRNA. The BMP-4 mRNA levels were normalized by the 18S rRNA level. (B, D) Images of GFP (left panel) and BMP-4-targeting molecular beacon (right panel) signals in control cells transfected with 100 nM of nonsilencing siRNA followed by adenovirus infection. The signal levels of MB1 (B) and MB8 (D) in the control cells corresponded well with the GFP intensity. (C, E) Images of GFP (left panel) and BMP-4-targeting molecular beacon (right panel) signals in cells transfected with 100 nM of BMP-4 siRNA followed by adenovirus infection. The same exposure times for imaging GFP and MB signals were used for all the experiments. Regardless of the GFP signal level, fluorescence signals from MB1 (C) and MB8 (E) almost diminished completely as a result of siRNA knockdown, indicating excellent beacon signal specificity. (F) Real-time PCR analysis suggesting that there is no antisense effect of BMP-4 targeting molecular beacons (MB1 and MB8) in live HDF cells. Cells were infected with virus for 2 days and then delivered with 1 μM of MB1 and MB8, respectively. Twenty-four hours after delivery, RNA was isolated from the cells. Bar graph shows the BMP-4 mRNA levels divided by that of control cells without any MB.