Input options for E-RNAi. Shown here is the input form of E-RNAi. The user is asked to choose a run option (de novo design, probe retrieval or probe evaluation) and to identify the organism for which to predict probe specificity. Users can also set the number of designed primers to be evaluated and of final results shown in the output. There is also an option to add SP6 or T7 promoter sequences to the designed primer pairs.

Analysis of genome-wide RNAi libraries. The Heidelberg/Boston RNAi library is evaluated for predicted efficiency and specificity of both individual siRNAs and dsRNAs. (A) Distribution of all siRNAs according to efficiency scores showed a mean score of 3.6. About 18.8% with an efficiency score ≥6 are considered efficient. (B) Over 90% of siRNAs are specific according to the specificity distribution. (C) The percentage of efficient siRNAs per gene and (D) The percentage of specific siRNAs for a single dsRNA.

Schematic representation of the program. The web application was implemented as Perl scripts and CGI modules that relied on BioPerl 1.5 (26). The data are in parts, stored in a relational MySQL database. Sequence homology searches are performed using BLAST (27). Primers to amplify PCR templates for dsRNAs are automatically designed using a local implementation of Primer3 (28). Genes and RNA templates are visualized in their genomic context using the Generic Genome Browser (29). Several data sources were integrated and are accessible through E-RNAi, including transcript and gene data obtained from Flybase [Drosophila, (30)] and Wormbase [C.elegans, (31)] and predesigned RNAi libraries (9,24). RNAi phenotypes were collected from Flybase, Wormbase and recent publications and assembled into a relational database that allows cross-species phenotype comparisons (unpublished data). In addition to the procedure shown here, the user can choose the option ‘probe retrieval’ to retrieve predesigned dsRNAs. When the ‘probe evaluation’ option is selected, the full-length input sequence is evaluated for efficiency and specificity parameters.

Output of the E-RNAi software. (A) De novo designed RNAi probes against the Rel gene are sorted by their overall score. In order to calculate this score, primer quality is weighted with 0.2, specificity with 0.25 and efficiency with 1. The table contains the length of each probe, the specificity and efficiency of each probe and the quality of primer pairs. (B) RNAi probes retrieved from RNAi libraries designed against the query are listed and evaluated. (C) Detailed information about each de novo designed probe is provided in the output page. A primer sequence with the SP6 promoter tag sequence (written in small letters), primer properties and probe sequence are shown. (D) De novo designed and retrieved probes are displayed on a GBrowse image at the bottom of the output page.

De novo design of optimized constructs for Rel and Mask. The coding regions of Rel (A) and Mask (D) were evaluated for specificity and efficiency in order to identify regions suitable for dsRNA design for RNAi experiments. All possibly efficient siRNAs in Rel (B) and Mask (E) are shown. Unspecific siRNAs in Rel (C) and Mask (F) are plotted to evaluate the possible off-target effects.