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Off-target effects of siRNA specific for GFP

Cordula Tschuch1, Angela Schulz1, Armin Pscherer1, Wiebke Werft2, Axel Benner2, Agnes Hotz-Wagenblatt3, Leticia Serra Barrionuevo1, Peter Lichter1* and Daniel Mertens14

Author Affiliations

1 Division of Molecular Genetics, German Cancer Research Center, Heidelberg, Germany

2 Division of Biostatistics, German Cancer Research Center, Heidelberg, Germany

3 Division of Molecular Biophysics, German Cancer Research Center, Heidelberg, Germany

4 Department of Internal Medicine III, University Hospital, Ulm, Germany

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BMC Molecular Biology 2008, 9:60  doi:10.1186/1471-2199-9-60

Published: 24 June 2008

Additional files

Additional file 1:

Additional genes down regulated by the GFP siRNA as measured by Real-Time PCR. Besides CYLD and SOAT, we measured by Real-Time PCR the mRNA levels of the off-target genes RAB21, NUDT3 and TCF4 that we identified in our microarray screen. Down modulation of the mRNA levels of these genes after transfection of GFP siRNA could be reproduced as for CYLD and SOAT. Shown are levels of off-target gene mRNA normalized to a mock-transfected control and to two housekeeping genes (see Methods).

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Additional file 2:

Genes deregulated after transfection with GFP siRNA.

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Additional file 3:

mRNAs deregulated by GFP siRNA contain short sequences perfectly matched to the sense or antisense strand of GFP siRNA. In order to define the background level of homologies to short sequence motifs, we i) shuffled the GFP siRNA sequence and ii) searched for homologies in genes that were either not deregulated or randomly picked. Shuffling of the GFP siRNA sequence was performed either with preserving the dinucleotide composition or without preservation of the dinucleotide composition (see Methods for details). The resulting shuffled GFP siRNA sequence was aligned to the set of genes that were deregulated by the proper GFP siRNA sequence in the profiling experiment ("shuffled dinucleotides preserved" and "shuffled dinucleotides not preserved"). For the negative control set of non-deregulated genes, we selected 207 genes that were least deregulated after transfection of GFP siRNA ("not dereg.A" and "not dereg. B") and 207 genes that were least deregulated in an unrelated profiling experiment ("diff.array", for details see Methods). We scored motifs of different lengths (ranging from >7 bp to >10 bp) that were homologous to the sequence of the transfected GFP siRNA. When comparing the sequence of GFP siRNA to sequences of the 207 significantly down regulated genes (grey bar), more genes with homology were found than in two sets of genes that were not regulated ("not dereg.A"; "not dereg.B") or taken from a non-related array ("diff.array"). With increasing length of homology, relatively more hits were scored for the proper, non-shuffled GFP siRNA in the set of actually deregulated genes as compared to all negative controls (bottom panels).

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Additional file 5:

Description of normalization of microarray data.

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Additional file 4:

qPCR primer sequences.

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