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. 2014 May 28;42(12):8049–8061. doi: 10.1093/nar/gku480

Figure 3.

Figure 3.

Off-target activity of different siPools. (A) HeLa cells were transfected with 10 nM siPool 60. To validate that the specific off-T siRNAs are present in the pools, Ago2 was immunoprecipitated from the lysates and passenger and guide strands of PolG off-T (left) or Scyl1 off-T (right) siRNAs were analyzed by northern blotting. As positive controls, 3 pmol of total siPools and 2.5% input material were used. (B) HeLa cells were transfected with 1, 3 or 10 nM siPool 15 #1, siPool 60 or specific off-T siRNAs directed against PolG (blue) or Scyl1 (green). Mad2 mRNA levels were measured by qPCR and normalized to GAPDH. Relative expression levels were calculated based on transfection of an unspecific control siRNA (neg. ctrl.). (C) Experiment was performed as described in (B). MAD2 protein levels were analyzed by western blotting 48 h after transfection. A specific MAD2 siRNA served as a positive control (lanes 9 and 10). Actin expression levels were used as loading controls (lower panels). (D) HeLa cells were transfected with 3 or 10 nM siRNA off-T or siPools containing 15, 30, 45 or 60 different siRNAs directed against PolG (blue) or Scyl1 (green). Off-target activity was analyzed using MAD2 3′-UTR controlling firefly-luciferase activity. Relative luciferase activity was calculated using the ratio of firefly/Renilla luciferase and via normalization to the corresponding ratios of the empty control vector.