Figure 2.

siPools efficiently knock down redundant gene family members. (A) HeLa cells were transfected with 3 or 10 nM concentrations of siPools targeting TNRC6A (‘A’, left panel), TNRC6B (‘B’, middle panel), TNRC6C (‘C’, right panel) or a combination of all three siPools (‘ABC’). Specific siRNAs against TNRC6A, TNRC6B or TNRC6C were transfected in similar concentrations. As a negative control for TNRC6 targeting siPools, an unspecific control siPool, and for siRNAs an unspecific control siRNA, was used. mRNA levels were measured by qPCR and normalized to GAPDH, and relative expression levels were calculated based on transfection of an unspecific control siPool or an unspecific control siRNA (ctrl.). (B) siPools specifically knock down individual family members. HeLa cells were transfected with 3 nM concentrations of siPools targeting TNRC6A (‘A’), TNRC6B (‘B’), TNRC6C (‘C’) or a combination of all three siPools (‘ABC’). mRNA levels of TNRC6A (left panel), TNRC6B (middle panel) or TNRC6C (right panel) were measured by qPCR and normalized to GAPDH. Relative expression levels were calculated based on transfection of an unspecific control siPool or an unspecific control siRNA (ctrl.). (C) Dual luciferase assay: HeLa cells were co-transfected with HMGA2 3′-UTR dual luciferase expression vector and with 3 or 10 nM siRNAs, or siPools targeting TNRC6A (T6A), TNRC6B (T6B), TNRC6C (T6C) or a combination of all siPools targeting TNRC6A, TNRC6B and TNRC6C (T6 ABC). As positive control we used a let-7a 2′OMe miRNA inhibitor which was normalized to an unspecific control 2′OMe inhibitor (ctrl.). Relative luciferase activity was calculated using the ratio for firefly/Renilla luciferase and via normalization to the corresponding ratios of an HMGA2 3′-UTR with mutated let-7a binding sites. All ratios were further normalized to a corresponding unspecific negative control (ctrl.) siPool or siRNA. (D) qPCR analysis of TNRC6A, TNRC6B and TNRC6C expression levels relative to GAPDH in HeLa cells.