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Open Access Research article

False negative rates in Drosophila cell-based RNAi screens: a case study

Matthew Booker13, Anastasia A Samsonova1, Young Kwon1, Ian Flockhart1, Stephanie E Mohr1 and Norbert Perrimon12*

Author Affiliations

1 Department of Genetics, Harvard Medical School, (77 Avenue Louis Pasteur), Boston, Massachusetts, (02115), USA

2 Howard Hughes Medical Institute, (77 Avenue Louis Pasteur), Boston, Massachusetts, (02115), USA

3 Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, (185 Meeting Street), Providence, Rhode Island, (02192), USA

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BMC Genomics 2011, 12:50  doi:10.1186/1471-2164-12-50

Published: 20 January 2011



High-throughput screening using RNAi is a powerful gene discovery method but is often complicated by false positive and false negative results. Whereas false positive results associated with RNAi reagents has been a matter of extensive study, the issue of false negatives has received less attention.


We performed a meta-analysis of several genome-wide, cell-based Drosophila RNAi screens, together with a more focused RNAi screen, and conclude that the rate of false negative results is at least 8%. Further, we demonstrate how knowledge of the cell transcriptome can be used to resolve ambiguous results and how the number of false negative results can be reduced by using multiple, independently-tested RNAi reagents per gene.


RNAi reagents that target the same gene do not always yield consistent results due to false positives and weak or ineffective reagents. False positive results can be partially minimized by filtering with transcriptome data. RNAi libraries with multiple reagents per gene also reduce false positive and false negative outcomes when inconsistent results are disambiguated carefully.