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

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© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 5. — Comparison of siPools with other available RNAi reagents. qPCR analysis of on- (left panels in (A) and (B)) and off-target (right panels in (A) and (B)) activities of various siRNA tools. HeLa cells were transfected with 1 or 3 nM siPool 15, siPool 60, single off-T siRNA, esiRNAs and four different low-complexity pools directed against PolG (A) or Scyl1 (B). mRNA levels were normalized to GAPDH and relative expression levels were calculated using a negative control siRNA. Low-complexity pool #4 served as a MAD2 off-target negative control. (C) HeLa cells were transfected with 1, 3 or 10 nM siRNA off-T, siPools with 15 or 60 different siRNAs and four different low-complexity pools directed against PolG (blue) or Scyl1 (green). Off-target activity was analyzed using a reporter system based on firefly-luciferase activity controlled by the MAD2 3′-UTR. Relative luciferase activity was calculated using the ratio of firefly/Renilla luciferase and via normalization to the corresponding ratios of the empty control vector. Low-complexity pool #4 served as a MAD2 off-target negative control.