University of Heidelberg

Cell and Molecular Biology

 

 

 

Technology Platform

 

RNAi Screening

We develop and apply high-throughput screening technologies to systematically survey the Drosophila genome for new functions. Our work focuses on automated RNA interference (RNAi) screenings to identify novel targets in cellular processes.

  • RNA interference

     

    RNA interference (RNAi) has emerged as a powerful tool to systematically dissect gene function by depleting transcripts through the introduction of homologous short or long double-stranded RNAs. Discovered in C. elegans and plants, RNAi is now also widely employed for gene-silencing studies in many model systems, including Drosophila and human cells. With the development and availability of genome-wide RNAi libraries in combination with high-throughput screening techniques, RNAi can be used to query genomes for a variety of cell or organism-based phenotypes.

     


    RNAi screening approaches
    (Picture from: Boutros M. and Ahringer, J. (2008). The art and design
    of genetic screens: RNA interference. Nature Rev. Genet., 9:554-566.)

     

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  • RNAi screening in Drosophila cells

     

    RNAi in cultured Drosophila cells can be triggered by adding in vitro generated double-stranded RNAs to the cell culture medium. Compared to screens in mammalian cells, Drosophila has the key advantages of relatively low genetic redundancy and high efficacy of RNAi reagents. Using this model organism our group was able to identify a number of new factors of Wnt, JAK/STAT, IMD and further conserved signaling pathways. Screening experiments can be performed genome-wide or for a small number of genes, for example those in specific functional groups. With cell-based assays we can measure a diverse set of phenotypes, ranging from homogenous cell viability readouts over alterations in reporter gene expression to high-content assays using automated microscopy.

     

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  • Publications

 

RNAi Screening Resources

Our group has long-standing experience in cell-based RNAi screening in Drosophila. We constantly develop and optimize screening reagents and methods. We developed and generated a novel genome-wide Drosophila RNAi library with an optimized design. Several genome-wide RNAi screens have been conducted so far using the second-generation library (Heidelberg 2). This library recently served to generate a genome-wide in vivo RNAi Drosophila library (VDRC KK library), enabling to perform cell-based and in vivo studies using the same RNAi constructs. The library is constantly updated according to new gene annotations and further developed in terms of specificity and efficiency.

 

Computational Tools

We developed a number of computational tools for the design and analysis of high-throughput screening experiments. These include software tools to design and evaluate RNAi reagents (E-RNAi, NEXT-RNAi), a user-friendly tool for statistical analysis of screening data and a comprehensive database for RNAi phenotypes.

  • web cellHTS2 - a web-based implementation of cellHTS2 for the statistical analysis of screening experiments

     

    High-throughput screens by RNAi generate large primary data sets which need to be analyzed and annotated to identify phenotypic hits. The web application, a front-end to the Bioconductor/R package cellHTS2, guides the user through all configuration steps required for analysis of single or multi-channel experiments. The web-application provides various options for standardization and normalization methods, annotation of data sets and a comprehensive HTML report of the screening data analysis, including a ranked hit list. Sessions can be saved and re-stored for later re-analysis.

     

    Reference: Pelz et al. (2010). web cellHTS2: a web-application for the analysis of high-throughput screening data. BMC Bioinformatics. 11:185. » Link to PubMed

     

    » Go to web cellHTS2

     

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  • GenomeRNAi - a comprehensive database for RNAi phenotypes from Drosophila and human screens

     

    The GenomeRNAi database contains phenotypes from published RNA interference (RNAi) screens in Drosophila melanogaster and Homo sapiens. The database connects observed phenotypes with annotations of targeted genes and information about the RNAi reagent used for the perturbation experiment. The availability of phenotypes from Drosophila and human screens also allows for phenotype searches across species. Besides reporting quantitative data from genome-scale screens, GenomeRNAi also enables reporting of data from microscopy experiments and curated phenotypes from published screens. In addition, the database provides an updated resource of RNAi reagents and their predicted quality that are available for the Drosophila and the human genome.

     

    Reference: Gilsdorf et al. (2010). GenomeRNAi: a database for cell-based RNAi phenotypes. 2009 update. Nucleic Acids Res., 38(Database issue):D448-52. » Link to PubMed

     

    » Go to GenomeRNAi

     

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  • E-RNAi - a web-based tool for the design of RNAi constructs

     

    For the correct assessment of phenotypes, a key issue remains the stringent quality control of long double-stranded RNAs (dsRNA) to calculate potential off-target effects that may obscure the phenotypic data. We have developed a web-based tool to evaluate and design optimized dsRNA constructs.

     

    Reference: Horn T. and Boutros M. (2010). E-RNAi: a web application for the multi-species design of RNAi reagents--2010 update. Nucleic Acids Res. 38(Web Server issue):W332-9. » Link to PubMed

     

    » Go to E-RNAi

     

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  • NEXT-RNAi - a software for the design and evaluation of genome-wide RNAi libraries

     

    NEXT-RNAi is a software for the design and evaluation of genome-wide RNAi libraries and performs all steps from the prediction of specific and efficient RNAi target sites to the visualization of designed reagents in their genomic context. The software enables the design and evaluation of siRNAs and long dsRNAs and was implemented in an organism-independent manner allowing designs for all sequenced and annotated genomes. It requires the minimal input of desired target sequences and an off-target database. NEXT-RNAi implements several methods to predict a reagents' quality and offers many special features such as the straight-forward design of independent RNAi reagents.

     

    Reference: Horn et al. (2010). Design and evaluation of genome-wide libraries for RNAi screens. Genome Biol. 11(6):R61. » Link to PubMed

     

    » Go to Next-RNAi

     

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General Information

  • Contact

     

    Department Head

    Prof. Dr. Michael Boutros

    Phone: +49 621 383 9650

    E-mail:

    Project Management

    Dr. Ulrike Hardeland

    Phone: +49 621 383 9648

    E-mail:

    Postal Address

    Heidelberg University, Medical Faculty Mannheim

    Department for Cell and Molecular Biology

    Ludolf-Krehl-Strasse 13-17

    68167 Mannheim

    Germany

     

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  • Enquiries about RNAi reagents and screening

     

    The second-generation Drosophila dsRNA library (Heidelberg 2) can be obtained from us in a pre-aliquoted ready-to-use form. We provide whole-genome RNAi libraries, sub-libraries or custom-made sets for selected targets. Please contact us for costs and further information.

    If you are interested in performing a screen with us, we would need to know certain information to determine whether we can perform such a screening experiment in collaboration. Most high-throughput screens have specific requirements with regards to assay development and screening setup.

    The form below helps us to understand the biological background of the screen and the technical requirement. Please email the form to . Please feel free to contact us and to discuss your screening idea.

     

    » Download Information request form

     

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Last updated: 15.05.2014