Visual Genome-Wide RNAi Screening to Identify Human Host Factors Required for Trypanosoma cruzi Infection
Figure 5
Confirming specificity of siRNA silencing.
To confirm specificity and exclude off-target effects of the siRNA pools used in the primary and secondary screens, the 4 different siRNA duplexes from the pool targeting each gene were separated and individually transfected to U2OS cells in 96-well plates following the same experimental conditions used for secondary screen. (A) Schematic representation of the four siRNAs targeting the calcium binding protein 2 (CABP2) mRNA sequence. bp = base pairs. (B) Pictures showing CABP2 gene silencing using the four different siRNAs depicted in (A). U2OS cells were transfected with each one of the siRNAs and then infected with T. cruzi parasites. Pictures a, b, c and d show a decrease in infection when compared to scrambled siRNA (bottom picture). All 13 genes tested were confirmed by at least two individual siRNAs, demonstrating that the infection inhibition seen in primary and secondary screens was not due to an off target effect. Scale bar represents 80 µm.