Figure 3.

Single-cell level quantification of RNAi on a microarray platform. The imaging and quantification of fluorescence in HeLa/d2eGFP cells plated on two representative siRNA spots (a negative control cat siRNA spot [A-F] and an experimental rh-egfp siRNA spot [G-L], 2.5 ng of siRNA per spot) on a microarray slide. To quantify the effect of the siRNAs on GFP silencing, green fluorescence DAPI-stained nuclei (A,G) and “watershed” boundaries (defined using the green fluorescence images, B and H) were used to locate and define the area for the cells (white) in each of the two images (C and I; see Supplemental Fig. A). These segmented images were used to measure single-cell parameters used in subsequent data analysis (Supplemental Material, data sets 1 and 2). (D,J) For each spot, the distribution of the median green fluorescence (after local background fluorescence subtraction) in every segmented cell was plotted. A cell was considered to be positive for eGFP green fluorescence when its median fluorescence intensity was >4 units in the green channel. The distributions of the median green fluorescence intensities obtained by microarray analysis (D,J) were compared with traditional flow cytometric measurements of Hela/d2eGFP cells (10,000 events) transfected with the same siRNAs (E,K). The vertical line indicates the estimated level of background fluorescence. The degree of gene silencing detected by flow cytometry was consistent with that detected by the RNAi microarray imaging system. Statistical analysis (F,L) indicated a decrease in the green fluorescence intensities when the data fromthe cat siRNA and an rh-egfp siRNA spot were compared, whereas other parameters, such as the total cell area and nuclear area were similar in the two spots.