TRBP is required for RNA interference mediated by shRNA or exogenous miRNA against EGFP in human cells. A) TRBP expression in human cells. Expression levels of TRBP was assessed in HeLa, Jurkat and U251MG cells using immunoblotting. The TRBP:Actin ratio calculated by densitometry is indicated below each lane. B, C, D) sh- and miRNA against EGFP are less active in astrocytes than in HeLa cells. HeLa (left) or astrocytes U251MG (right) cells were transfected with 1 μg of EGFP-C1 and 4 μg of ns or anti-EGFP sh- or miRNAs as indicated. B) Cellular fluorescence of live cells was monitored on an inverted microscope and a representative picture is shown. Percentages reflect knock-down of EGFP expression as calculated by mean luminosity intensity. C) Cells were analyzed by FACS. Reporter expression is calculated as the percentage of EGFP expression in the presence of sh or miRNA compared to ns normalized to 100%. Values are means of four independent experiments ± SEM. D) Total cell extracts were analyzed by immunoblot for EGFP and actin expression. Percent decrease of EGFP signal was calculated by densitometry analysis.
Daniels et al. BMC Molecular Biology 2009 10:38 doi:10.1186/1471-2199-10-38