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. 2009 Jan 5;4(1):e4118. doi: 10.1371/journal.pone.0004118

Figure 2. siRNA's efficiently suppresses gB and Ori expression at the mRNA and protein level.

Figure 2

(A). Relative qRT-PCR. RK13 cells were transfected with 37.5 pmol sigB3, 75 pmol siOri2, 75 pmol siLuc or not transfected. Cells were infected 14 h later with 500 PFU of rAb4Δgp2 and at 12 h p.i., RNA was extracted from infected cells. RT-PCR was used to determine relative levels of gB or Ori (white bars) or EHV-1 IR6 (grey bars) mRNA, using rabbit β-actin as the endogenous housekeeping gene. Asterisks indicate statistically significant differences (p<0.05). (B). Western blot analysis. RK13 cells were either not transfected or transfected with 75 pmol of the control siRNA siLuc or 75 pmol sigB3. Cells were infected 14 h later with 500 PFU of rAb4Δgp2. At 24 h p.i., cell lysates were prepared and analyzed by SDS-PAGE under reducing conditions. Anti-gB mAb 3F6 (1/500) and anti-β-actin (1/5000), followed by anti-mouse IgG peroxidase (1/7500) were used. Cell lysates from non-infected RK13 cells were included as a control.