Figure 3.

Schematic diagram of the hierarchical RNAi analysis for identifying genes that are involved in a specific phenotype. R = {r₁, r₂, …, r_k} is the set of qualified siRNAs that were selected by our modified algorithm. The candidate-genes in each ellipse are the target genes of each available siRNA r_i. In the first-round RNAi analysis, we can quickly determine which candidate-genes could be related to the target phenotype based on the RNAi experiment results. The green ellipse represents the target phenotype that was affected in the RNAi experiment with r_i, whereas the blue ellipse indicates that the target phenotype was not affected. In the second-round RNAi analysis, we further examined the candidate-genes in the green ellipses to precisely identify the related genes. However, the complementary strand of the selected siRNA, r^′_i, can also be loaded into the RISC, and it could facilitate RNAi. A gene that contains a subsequence that contains no more than d_T mismatches to r^′_i would be regarded as the possible target gene of r^′_i, which is defined as X_i. To precisely identify the associated gene from the genes that are affected by r_i and r^′_i, we would silence those genes one-on-one to identify which genes were definitively associated with the target phenotype.