Down-regulation of CD24 expression by siRNA is associated with reduced expression of selected genes encoding tight junction proteins. Data shown are representative of three independent experiments in each case. (A) Showing down-regulation of mRNA for CD24 in H413 clone-1 cells with two different inserts compared to control plasmid following 30 cycles of RT-PCR. (B) Reverse transcription (RT)-PCR array (30 cycles) showing down-regulated gene expression of tight junction proteins for CD24 siRNA insert-1 silenced H413 clone-1 cells and compared to the cells transfected with control plasmid. Bands were visualized on a 2% agarose gel after staining with ethidium bromide. (C) Showing weaker suppression of expression of genes encoding tight junction proteins following transfection with the second CD24 siRNA insert that was less effective in suppressing CD24 expression (Fig. 7A). (D, E) Showing un-silenced CD24 following transfection by siRNA-3 (E) had no detectable effect on expression of genes encoding tight junction proteins as compared to plasmid control (D). [β-actin controls in D also apply to E]. (F) Bar graph showing RT-PCR arrays (30 cycles) showing gene expression in CD24 siRNA-1 silenced H413 clone-1 cells compared to plasmid control. Data are mean values from three independent experiments (± s.e.m.). The values for each experiment were normalized against the expression of β-actin (assigned as 100) and compared with the control sample by paired t-test. * Refers to P < 0.05; ND = not detected; five tight junction genes showed significant reductions that were confirmed by real-time quantitative RT-PCR analysis in Table 2. Expression of genes encoding claudin-3 and par-6 was not detected in either CD24 siRNA silenced cells or control plasmid culture samples by real-time quantitative RT-PCR. All experiments were performed 48 h after transfection.