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Genome-wide resources of endoribonuclease-prepared short interfering RNAs for specific loss-of-function studies

Abstract

RNA interference (RNAi) has become an important technique for loss-of-gene-function studies in mammalian cells. To achieve reliable results in an RNAi experiment, efficient and specific silencing triggers are required. Here we present genome-wide data sets for the production of endoribonuclease-prepared short interfering RNAs (esiRNAs) for human, mouse and rat. We used an algorithm to predict the optimal region for esiRNA synthesis for every protein-coding gene of these three species. We created a database, RiDDLE, for retrieval of target sequences and primer information. To test this in silico resource experimentally, we generated 16,242 esiRNAs that can be used for RNAi screening in human cells. Comparative analyses with chemically synthesized siRNAs demonstrated a high silencing efficacy of esiRNAs and a 12-fold reduction of downregulated off-target transcripts as detected by microarray analysis. Hence, the presented esiRNA libraries offer an efficient, cost-effective and specific alternative to presently available mammalian RNAi resources.

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Figure 1: In silico prediction of esiRNA template sequences.
Figure 2: Gene-centric information in the RiDDLE database.
Figure 3: Generation of a human genome-wide esiRNA library.
Figure 4: Silencing efficacy of esiRNAs.
Figure 5: Silencing specificity of esiRNAs.

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Acknowledgements

We thank Eberhard Krausz (Technology Development Studio, Max Planck Institute for Molecular Cell Biology and Genetics) for providing robotics support, and the Rosetta Gene Expression Laboratory for microarray hybridizations. This work was supported by the EU grants “FunGenES” (LSHG-CT-2003-503494), “Mitocheck” (LSHG-CT-2004-503464), by BMBF grant PTJ-BIO/0313130, the NGFN2 grant SMP-RNAi (01GR0402) and the Max Planck Society.

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Authors and Affiliations

Authors

Contributions

R.K., A.K.H., M.S., M.T., G.P., K.F. and A.C. generated the human esiRNA library; V.S. and B.H. performed the in silico analyses; H.G. and J.W. performed automation; K.K. generated LIMS; E.R. and B.K. generated esiRNAs; C.F. performed QPCRs; C.S. and B.S. performed and analyzed QPCRs; J.G., J.S., J.B. and A.L.J. performed and analyzed microarray studies; P.S.L. analyzed microarray data; R.K. and F.B. designed and analyzed the experiments; R.K., V.S., A.L.J., B.H. and F.B. wrote the paper.

Corresponding authors

Correspondence to Bianca Habermann or Frank Buchholz.

Ethics declarations

Competing interests

V.S. and B.H. work for Scionics Computer Innovation.

E.R. and B.K. work for the RZPD.

C.F., C.S. and B.S. work for Cenix Bioscience.

J.G., J.S., J.B., P.S.L. and A.L.J. work for Rosetta Inpharmatics.

Supplementary information

Supplementary Fig. 1

Correlation of silencing efficacy between esiRNAs and siRNAs. (PDF 452 kb)

Supplementary Fig. 2

No induction of interferon response genes by esiRNA and siRNA. (PDF 600 kb)

Supplementary Fig. 3

Comparative analysis of off-target regulation. (PDF 319 kb)

Supplementary Table 1

Primer sequences used to generate esiRNAs. (PDF 13 kb)

Supplementary Table 2

Sequences of employed siRNAs. (PDF 11 kb)

Supplementary Table 3

Primers employed for QPCRs. (PDF 10 kb)

Supplementary Methods (PDF 11 kb)

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Kittler, R., Surendranath, V., Heninger, AK. et al. Genome-wide resources of endoribonuclease-prepared short interfering RNAs for specific loss-of-function studies. Nat Methods 4, 337–344 (2007). https://doi.org/10.1038/nmeth1025

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