RNAi Experiments in D. melanogaster: Solutions to the Overlooked Problem of Off-Targets Shared by Independent dsRNAs

Seinen, Erwin; Burgerhof, Johannes G. M.; Jansen, Ritsert C.; Sibon, Ody C. M.

doi:10.1371/journal.pone.0013119

Cited by 17 publications

(22 citation statements)

References 25 publications

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“…One common problem while using RNAi transgenes is off-targeting (Seinen et al, 2010, 2011). In order to confirm that the observed phenotypes are due to downregulating of fumble and not to secondary effects of the specific RNAi transgene, we tested a different UAS-RNAi transgene against fbl .…”

Section: Resultsmentioning

confidence: 99%

A new in vivo model of pantothenate kinase-associated neurodegeneration reveals a surprising role for transcriptional regulation in pathogenesis

Pandey¹,

Turm²,

Bekenstein³

et al. 2013

Front. Cell. Neurosci.

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Pantothenate Kinase-Associated Neurodegeneration (PKAN) is a neurodegenerative disorder with a poorly understood molecular mechanism. It is caused by mutations in Pantothenate Kinase, the first enzyme in the Coenzyme A (CoA) biosynthetic pathway. Here, we developed a Drosophila model of PKAN (tim-fbl flies) that allows us to continuously monitor the modeled disease in the brain. In tim-fbl flies, downregulation of fumble, the Drosophila PanK homologue in the cells containing a circadian clock results in characteristic features of PKAN such as developmental lethality, hypersensitivity to oxidative stress, and diminished life span. Despite quasi-normal circadian transcriptional rhythms, tim-fbl flies display brain-specific aberrant circadian locomotor rhythms, and a unique transcriptional signature. Comparison with expression data from flies exposed to paraquat demonstrates that, as previously suggested, pathways others than oxidative stress are affected by PANK downregulation. Surprisingly we found a significant decrease in the expression of key components of the photoreceptor recycling pathways, which could lead to retinal degeneration, a hallmark of PKAN. Importantly, these defects are not accompanied by changes in structural components in eye genes suggesting that changes in gene expression in the eye precede and may cause the retinal degeneration. Indeed tim-fbl flies have diminished response to light transitions, and their altered day/night patterns of activity demonstrates defects in light perception. This suggest that retinal lesions are not solely due to oxidative stress and demonstrates a role for the transcriptional response to CoA deficiency underlying the defects observed in dPanK deficient flies. Moreover, in the present study we developed a new fly model that can be applied to other diseases and that allows the assessment of neurodegeneration in the brains of living flies.

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Section: Resultsmentioning

confidence: 99%

A new in vivo model of pantothenate kinase-associated neurodegeneration reveals a surprising role for transcriptional regulation in pathogenesis

Pandey¹,

Turm²,

Bekenstein³

et al. 2013

Front. Cell. Neurosci.

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“…After this filtering step, we had around 200 to 300 cells per knock-down. A second filtering step concerns siRNA off-targets [ 25 ]. We sought to avoid confounding by this effect and therefore selected only genes with low predicted off-target effects as described in ‘siRNA filtering for off-targets’ of S1 Text .…”

Section: Resultsmentioning

confidence: 99%

NEMix: Single-cell Nested Effects Models for Probabilistic Pathway Stimulation

et al. 2015

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Nested effects models have been used successfully for learning subcellular networks from high-dimensional perturbation effects that result from RNA interference (RNAi) experiments. Here, we further develop the basic nested effects model using high-content single-cell imaging data from RNAi screens of cultured cells infected with human rhinovirus. RNAi screens with single-cell readouts are becoming increasingly common, and they often reveal high cell-to-cell variation. As a consequence of this cellular heterogeneity, knock-downs result in variable effects among cells and lead to weak average phenotypes on the cell population level. To address this confounding factor in network inference, we explicitly model the stimulation status of a signaling pathway in individual cells. We extend the framework of nested effects models to probabilistic combinatorial knock-downs and propose NEMix, a nested effects mixture model that accounts for unobserved pathway activation. We analyzed the identifiability of NEMix and developed a parameter inference scheme based on the Expectation Maximization algorithm. In an extensive simulation study, we show that NEMix improves learning of pathway structures over classical NEMs significantly in the presence of hidden pathway stimulation. We applied our model to single-cell imaging data from RNAi screens monitoring human rhinovirus infection, where limited infection efficiency of the assay results in uncertain pathway stimulation. Using a subset of genes with known interactions, we show that the inferred NEMix network has high accuracy and outperforms the classical nested effects model without hidden pathway activity. NEMix is implemented as part of the R/Bioconductor package ‘nem’ and available at www.cbg.ethz.ch/software/NEMix.

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“…due to a lack of robust knockdown. Others have previously reviewed or presented approaches to detecting and limiting the impact of false discovery, which include improvements to reagent design, improvements to statistical analyses, and comparison of primary ‘hits’ with transcriptomics data [61] [2,36,43,62–65]. For plate-reader screens, it can be useful to eliminate hits corresponding to severely reduced cell viability prior to further analysis, as exemplified in a signaling study [33].…”

Section: Limiting False Discovery In Cell-based or In Vivo Rnai Scmentioning

confidence: 99%

RNAi screening in Drosophila cells and in vivo

Mohr

2014

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RNAi Experiments in D. melanogaster: Solutions to the Overlooked Problem of Off-Targets Shared by Independent dsRNAs

Cited by 17 publications

References 25 publications

A new in vivo model of pantothenate kinase-associated neurodegeneration reveals a surprising role for transcriptional regulation in pathogenesis

A new in vivo model of pantothenate kinase-associated neurodegeneration reveals a surprising role for transcriptional regulation in pathogenesis

NEMix: Single-cell Nested Effects Models for Probabilistic Pathway Stimulation

RNAi screening in Drosophila cells and in vivo

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