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A case study of the reproducibility of transcriptional reporter cell-based RNAi screens in Drosophila

Ramanuj DasGupta1*, Kent Nybakken2, Matthew Booker3, Bernard Mathey-Prevot3, Foster Gonsalves1, Binita Changkakoty1 and Norbert Perrimon34

Author Affiliations

1 New York University School of Medicine/Cancer Institute, Department of Pharmacology, First Avenue, New York, NY 10016, USA

2 Boston Biomedical Research Institute, 64 Grove Street, Watertown, MA, 02472, USA

3 Department of Genetics, Harvard Medical School, Avenue Louis Pasteur, Boston, MA 02115, USA

4 Howard Hughes Medical Institute, Harvard Medical School, Avenue Louis Pasteur, Boston, MA 02115, USA

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Genome Biology 2007, 8:R203  doi:10.1186/gb-2007-8-9-r203

Published: 28 September 2007

Additional files

Additional data file 1:

The log-ratio of normalized luciferase units of experimental dsRNA (Nexp) with that of GFP dsRNA (Ngfp) is listed. Experiments were performed twice in triplicates (six data points for each gene tested). A consistent increase or decrease of at least 30% of the reporter activity with respect to the average of multiple negative controls (GFP dsRNA) was considered as a positive hit. Validation information for a second dsRNA is also provided for the genes that could be validated by the first amplicon.

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Additional data file 2:

Genes name and CG# provided for those dsRNAs that were reported to have multiple potential OTs in the previously published Wnt/wg screen of DasGupta et al. [3], but still pass the validation test with DRSV-v dsRNAs (first column). Also listed are genes/CG# representing dsRNAs that represent unique amplicons in the DasGupta et al. screen and still pass with validation dsRNAs of the DRSC-v library (column 2). Note that several dsRNAs of the DRSC1.0 library that were thought to have OTEs could be re-validated using unique DRSC-v amplicons. Moreover, not all unique dsRNAs of the DRSC1.0 library had reproducible effects on the modulation of the Wg reporter activity when a corresponding unique validation dsRNAs (DRSC-v) was used.

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Additional data file 3:

The number of potential off-targets calculated for the amplicon that was identified in the original Hh screen, based on a 19 bp window, is listed once for each gene. The average fractional change in reporter activity compared to GFP dsRNA controls (listed at the bottom) are presented, with scores between -0.25 and -0.50 highlighted in yellow, scores less than -0.50 highlighted in orange, and scores greater than or equal to + 0.50 highlighted in blue. At the bottom of the list, scores for GFP, Ci, Smo, and th dsRNA controls that were included in the assay plates are also indicated.

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Additional data file 4:

Drosophila Cl8 cells were transfected with validation dsRNAs and the Wg-responsive luciferase reporter (dTF12). The log ratio of normalized luciferase units were computed as log(N-drsc_v/N-gfp) and plotted on a bar graph. Candidate negative and positive regulators are represented by negative and positive log ratios respectively, as compared to the GFP dsRNA control. Since the ratio of N_gfp/N_gfp is 1, the log ratio for gfp dsRNA control is zero.

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