Relative luciferase expression showing the RNAi response in human fibroblasts. Primary human skin fibroblasts from two fragile × patients (Fra-X1 and 2) and a normal control person with a normal karyotype were transfected with the plasmid pGL3-basic (Promega) encoding luciferase and siRNA against the luciferase (GL3 siRNA, MWG-Biotech). GL3 siRNA targets the luciferase sequence 5'-CUUACGCUGAGUACUUCGATT -3' . As control for the transfection frequency, cells were also transfected with the plasmid pCMVβ (Clontech) encoding beta-galactosidase. Luciferase and beta-galactosidase activities were measured one and two days after transfection using kits from Pierce and Promega, respectively. Transfections were performed in triplicates using X-tremeGeNE siRNA Transfection Reagent as suggested by the manufacturer (Roche). Subconfluent cells in 25 cm2 tissue culture flasks were transfected with a mixture of 100 μl transfection reagent diluted in Opti-MEM together with 6 μg of each of the reporter plasmids and 2 μg of the siRNA oligonucleotides. The primary fibroblasts were obtained from 2 patients with a trinucleotide expansion leading to a full mutation in the FMR1 gene, and, thus, no FMRP protein detectable using western blotting (data not shown). A negative control siRNA with no significant homology to any known gene sequences from mouse, rat, or human was also purchased from MWG-Biotech. Shown on the Y-axes are the luciferase expression divided by the beta-galactosidase expression, setting the ratio to 100 for cells transfected without siRNA. Error bars: Standard deviation. The experiments comply with the National regulations on cells from approved biobanks. The primary human fibroblasts were cultured according to standard procedures and used in early passages.