Figure 4.

Assessment of the sensitivity and specificity of the RNAi microarray platform. (A) An analysis of eGFP expression (median green fluorescence intensity) in HeLa/d2eGFP cells over time following exposure to either arrayed egfp (black bars) or negative control (cat) siRNAs (gray bars; 2.5 ng siRNA per spot). The inhibition of eGFP expression by arrayed egfp siRNAs (black bars) is dose-dependent (0.25 ng, 1 ng, or 2.5 ng of siRNAs), and this effect can be observed over time. (B) 24 h; (C) 72 h. No change was seen in the fluorescence intensity of Hela/d2eGFP cells exposed to the negative control cat siRNAs (gray bars). (D) Cells exposed to arrayed siRNAs (negative control, gray bars; experimental siRNAs, black bars) were of the same size and were otherwise comparable; see Supplemental Material for additional statistics. Three different siRNAs against egfp were compared for their ability to inhibit eGFP expression using RNAi microarray (2.5 ng siRNA) (E) and flow cytometry (2 μg siRNA was transfected using Lipofectin into ∼2 × 10⁵ HeLa/d2eGFP cells) (F). (A-E) The average median green fluorescence of triplicate siRNA spots is plotted with the error bar representing 1 SD; (F) the average geometric mean green fluorescence of three independent transfections is plotted with the error bar representing 1 SD.