Classic Papers Series: Lin et al. show RNAi screen dominated by seed effects

Classic Papers Series: Lin et al. show RNAi screen dominated by seed effects

Over the coming months, we will highlight a number of seminal papers in the RNAi field. The first such paper is from 2005 by Lin et al. of Abbott Laboratories, who showed that the top hits from their RNAi screen were due to seed-based off-target effects, rather than the intended (and at that time, rather expected) on-target effect. The authors screened 507 human kinases with 1 siRNA per gene, using a HIF-1 reporter assay to identify genes regulating hypoxia-induced HIF-1 response. In…

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Tips for optimizing RNA affinity purification

Tips for optimizing RNA affinity purification

RNA affinity purification (RAP) experiments enable the isolation and analysis of interacting molecules with an RNA of interest. Often performed to gain insight into RNA function, it is gaining popularity in the study of lncRNAs but can also be applied to coding RNAs. The general workflow involves preserving nucleic acid and protein interactions with a cross-linking reagent, followed by lysis and sonication to shear nucleic acids to sizes amenable for pulldown. Biotin-labelled DNA probes (which we offer, see raPOOLs) are added to the…

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The limits of chemical modification

The limits of chemical modification

In addition to potential reductions in on-target efficiency, chemically modifying siRNAs will not necessarily eliminate seed-based off-target effects. Rasmussen et al. found that a chemically-modified siRNA can still have substantial seed effects. They examined the expression data for 3 siRNAs from Jackson et al. and showed that for one of them the seed is still active following chemical modification. The algorithm of Rasmussen et al. (cWords) looks through a ranked list of 3′ UTR sequences (in this case, ranked by deregulation as measured…

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Deconvoluted SMARTpools are like a box of chocolates

Deconvoluted SMARTpools are like a box of chocolates

… you never know what you’re going to get! Falkenberg et al. (2014) performed a synthetic lethal  RNAi screen to identify genes which, when knocked down in combination with drug treatment, induced apoptosis in drug-resistant cells. The first screening pass covered over 18,000 protein-coding genes using Dharmacon’s siGENOME, probably the most widely used library for genome-wide RNAi screens. siGENOME is based on low-complexity pooling, with 4 siRNAs pooled per gene.  In the first pass, hits are identified based on the pooled siRNA…

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Intronic off-target effects with antisense oligos

Intronic off-target effects with antisense oligos

Undecided on whether to use silencing RNAs (siRNAs) or antisense oligos (ASOs) for your RNA interference experiments? Read on. ASOs are single-stranded DNA oligonucleotides which downregulate specific RNA by hybridizing with it, forming a heteroduplex recognizable by RNase H1. RNase H1 is found in the nucleus, hence ASOs are often used to target nuclear-localized RNAs such as non-coding RNA. A recent paper however highlighted that since RNase H1 is found in the nucleus, intronic sequences may also fall prey to ASO-mediated silencing,…

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Simplicity is the ultimate sophistication

Simplicity is the ultimate sophistication

The beauty of the siPOOL strategy is its simplicity. In this presentation from the  (relatively) early days of Apple, Steve Jobs says that his company’s goal is to serve the one-on-one relationship between a user and his/her computer.   Similarly, siPOOLs, are designed to serve the one-on-one relationship between a scientist and his/her RNAi results. By providing an interpretable result without the need for extensive follow-up work and off-target corrections, siPOOLs make it possible for a scientist to use a…

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Seed effects persist in hyperdimensional space

Seed effects persist in hyperdimensional space

Work from the Carpenter lab suggests that attempts to shake seed-based off-targets by going to  ‘phenotypic hyperspace’ will not work. They performed a high-content assay with 315 shRNAs covering 41 genes.  A 1301-dimensional profile was created for each well, and compressed to 205 principal components that captured  99% of the variance. The hope would be that by examining a wider phenotypic space, the gene-specific effects of RNAi reagents would become more prominent. However, the profiles between shRNAs targeting the same…

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Knocking out the phenotype

Knocking out the phenotype

Consistent with the work of Rossi et al. (discussed previously),  another recent paper shows a lack of phenotypic response when knocking out a gene that gives a phenotypic response when knocked down. Knocking out klf2a does not result in any discernible difference from wild-type (whereas knock-down has been shown to produce a range of cardiovascular phenotypes). The authors conclude: In summary, our work shows that even in the face of clear evidence of a potentially disruptive mutation induced in a gene…

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Genetic compensation

Genetic compensation

Recent work by Rossi et al. shows that an unintended consequence of gene knockout may be genetic compensation that mitigates phenotypes. Knockdown in zebrafish of egfl7, an endothelial extracellular gene, causes severe vascular defects: However, following knockout of eglf7, there was no visible effect on vascular development, even after application of the knockdown reagent (demonstrating that the knockdown phenotype was not due to an off-target and that the knockout’s normal vascular development was not due some minor levels of egfl7): The authors found…

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Russian roulette

Russian roulette

Which bases should you choose for the seed region of a single siRNA? It’s like Russian Roulette on full-automatic, where a specific seed will result in dozens or hundreds of down-regulated genes. If you’re lucky, none of the off-targets results in a false-positive phenotype.   But odds are that you won’t be so lucky. A recent paper suggests that taking single siRNA drugs may be closer to real Russian Roulette than anyone would hope. The authors show that an siRNA designed to knock down…

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