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1.  cSSMD: assessing collective activity for addressing off-target effects in genome-scale RNA interference screens 
Bioinformatics  2011;27(20):2775-2781.
Motivation: Off-target activity commonly exists in RNA interference (RNAi) screens and often generates false positives. Existing analytic methods for addressing the off-target effects are demonstrably inadequate in RNAi confirmatory screens.
Results: Here, we present an analytic method assessing the collective activity of multiple short interfering RNAs (siRNAs) targeting a gene. Using this method, we can not only reduce the impact of off-target activities, but also evaluate the specific effect of an siRNA, thus providing information about potential off-target effects. Using in-house RNAi screens, we demonstrate that our method obtains more reasonable and sensible results than current methods such as the redundant siRNA activity (RSA) method, the RNAi gene enrichment ranking (RIGER) method, the frequency approach and the t-test.
Supplementary information: Supplementary data are available at Bioinformatics online.
PMCID: PMC3202303  PMID: 21846737
2.  A Lentivirus-Mediated Genetic Screen Identifies Dihydrofolate Reductase (DHFR) as a Modulator of β-Catenin/GSK3 Signaling 
PLoS ONE  2009;4(9):e6892.
The multi-protein β-catenin destruction complex tightly regulates β-catenin protein levels by shuttling β-catenin to the proteasome. Glycogen synthase kinase 3β (GSK3β), a key serine/threonine kinase in the destruction complex, is responsible for several phosphorylation events that mark β-catenin for ubiquitination and subsequent degradation. Because modulation of both β-catenin and GSK3β activity may have important implications for treating disease, a complete understanding of the mechanisms that regulate the β-catenin/GSK3β interaction is warranted. We screened an arrayed lentivirus library expressing small hairpin RNAs (shRNAs) targeting 5,201 human druggable genes for silencing events that activate a β-catenin pathway reporter (BAR) in synergy with 6-bromoindirubin-3′oxime (BIO), a specific inhibitor of GSK3β. Top screen hits included shRNAs targeting dihydrofolate reductase (DHFR), the target of the anti-inflammatory compound methotrexate. Exposure of cells to BIO plus methotrexate resulted in potent synergistic activation of BAR activity, reduction of β-catenin phosphorylation at GSK3-specific sites, and accumulation of nuclear β-catenin. Furthermore, the observed synergy correlated with inhibitory phosphorylation of GSK3β and was neutralized upon inhibition of phosphatidyl inositol 3-kinase (PI3K). Linking these observations to inflammation, we also observed synergistic inhibition of lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines (TNFα, IL-6, and IL-12), and increased production of the anti-inflammatory cytokine IL-10 in peripheral blood mononuclear cells exposed to GSK3 inhibitors and methotrexate. Our data establish DHFR as a novel modulator of β-catenin and GSK3 signaling and raise several implications for clinical use of combined methotrexate and GSK3 inhibitors as treatment for inflammatory disease.
PMCID: PMC2731218  PMID: 19727391
3.  Hit selection with false discovery rate control in genome-scale RNAi screens 
Nucleic Acids Research  2008;36(14):4667-4679.
RNA interference (RNAi) is a modality in which small double-stranded RNA molecules (siRNAs) designed to lead to the degradation of specific mRNAs are introduced into cells or organisms. siRNA libraries have been developed in which siRNAs targeting virtually every gene in the human genome are designed, synthesized and are presented for introduction into cells by transfection in a microtiter plate array. These siRNAs can then be transfected into cells using high-throughput screening (HTS) methodologies. The goal of RNAi HTS is to identify a set of siRNAs that inhibit or activate defined cellular phenotypes. The commonly used analysis methods including median ± kMAD have issues about error rates in multiple hypothesis testing and plate-wise versus experiment-wise analysis. We propose a methodology based on a Bayesian framework to address these issues. Our approach allows for sharing of information across plates in a plate-wise analysis, which obviates the need for choosing either a plate-wise or experimental-wise analysis. The proposed approach incorporates information from reliable controls to achieve a higher power and a balance between the contribution from the samples and control wells. Our approach provides false discovery rate (FDR) control to address multiple testing issues and it is robust to outliers.
PMCID: PMC2504311  PMID: 18628291
4.  Genome-wide screens for effective siRNAs through assessing the size of siRNA effects 
BMC Research Notes  2008;1:33.
RNA interference (RNAi) has been seen as a revolution in functional genomics and system biology. Genome-wide RNAi research relies on the development of RNAi high-throughput screening (HTS) assays. One of the most fundamental challenges in RNAi HTS is to glean biological significance from mounds of data, which relies on the development of effective analytic methods for selecting effective small interfering RNAs (siRNAs).
Based on a recently proposed parameter, strictly standardized mean difference (SSMD), I propose an analytic method for genome-wide screens of effective siRNAs through assessing and testing the size of siRNA effects. Central to this method is the capability of SSMD in quantifying siRNA effects. This method has relied on normal approximation, which works only in the primary screens but not in the confirmatory screens. In this paper, I explore the non-central t-distribution property of SSMD estimates and use this property to extend the SSMD-based method so that it works effectively in either primary or confirmatory screens as well as in any HTS screens with or without replicates. The SSMD-based method maintains a balanced control of false positives and false negatives.
The central interest in genome-wide RNAi research is the selection of effective siRNAs which relies on the development of analytic methods to measure the size of siRNA effects. The new analytic method for hit selection provided in this paper offers a good analytic tool for selecting effective siRNAs, better than current analytic methods, and thus may have broad utility in genome-wide RNAi research.
PMCID: PMC2526086  PMID: 18710486

Results 1-4 (4)