High-throughput RNAi screening to dissect cellular pathways: A how-to guide

RNA interference (RNAi) has become a powerful tool to dissect cellular pathways and characterize gene functions. The availability of genome-wide RNAi libraries for various model organisms and mammalian cells has enabled high-throughput RNAi screenings. These RNAi screens successfully identified key components that had previously been missed in classical forward genetic screening approaches and allowed the assessment of combined loss-of-function phenotypes. Crucially, the quality of RNAi screening results depends on quantitative assays and the choice of the right biological context. In this review, we provide an overview on the design and application of high-throughput RNAi screens as well as data analysis and candidate validation strategies.     

A dynamic perspective of RNAi library development

Shortly after the dissertation of the mechanism of RNA interference (RNAi), various RNAi libraries for invertebrates, plants or mammals that enable loss-of-function genetic screens on a genome-wide scale have been developed. Joint academic and industrial effort has led to the commercial launch of many of these libraries and this field is expected to continuously evolve at incredible speed. This article comparatively reviews the principles and applications of different RNAi libraries: from earlier synthetic to recent lentiviral RNAi libraries. The unique properties and limitations of each library will be important references for instigators to choose a particular library for their specific application.     

Computational detection and suppression of sequence-specific off-target phenotypes from whole genome RNAi screens

A challenge for large-scale siRNA loss-of-function studies is the biological pleiotropy resulting from multiple modes of action of siRNA reagents. A major confounding feature of these reagents is the microRNA-like translational quelling resulting from short regions of oligonucleotide complementarity to many different messenger RNAs. We developed a computational approach, deconvolution analysis of RNAi screening data, for automated quantitation of off-target effects in RNAi screening data sets. Substantial reduction of off-target rates was experimentally validated in five distinct biological screens across different genome-wide siRNA libraries. A public-access graphical-user-interface has been constructed to facilitate application of this algorithm.     

RNA干扰文库及其在功能基因组学研究中的应用

随着人类基因组大规模测序的完成,下一步的挑战是了解每一个基因的功能 . RNA 干扰文库为大规模基因功能筛选提供了可能 . 虽然用于线虫等模式生物的 RNAi 文库,已经证明是大规模基因功能筛选的有效方法,但这些文库不能用于高等动物的细胞 . 自 2003 年以来,用于人的细胞和哺乳动物细胞的 RNAi 文库取得了突破,相继出现构建已知基因 RNAi 文库和构建随机 RNAi 文库的报道,并成功地应用于大规模基因功能的筛选 . RNAi 文库作为一种简单、高效、大规模、高通量的功能基因组学研究的工具,将在基因功能研究、发现新的药物靶基因、发现疾病相关基因等方面有广阔的应用前景 .     

High-throughput RNAi screening in mammalian cells with esiRNAs

The development of advanced functional genomic tools has paved the way for systematic investigations of biological processes in health and disease. In particular, the implementation of RNA interference (RNAi) as a genome-wide, loss-of-function screening tool has enabled scientists to probe the role for every gene in cellular assays and many new factors for various processes have been discovered employing RNAi screens in recent years. However, the results also demonstrate the complexity of biological systems and indicate that we are still a long way from understanding functional networks in depth. Nevertheless, RNAi screens present a powerful method to interrogate gene function in high-throughput and different methods to elicit RNAi in mammalian cells have been developed. Here, we describe steps that should be considered when planning an RNAi screen employing endoribonuclease prepared (e)siRNAs. We provide useful information on how to implement the screen and analyze the results. Furthermore, we discuss strategies for hit validation and present an outline on how to follow-up on verified hits to gain a molecular understanding of the underlying phenotypes.     

A primer on using pooled shRNA libraries for functional genomic screens

The discovery of RNA interference (RNAi) has revolutionized genetic analysis in mammalian cells. Loss-of-function RNAi screens enable rapid, functional annotation of the genome. Of the various RNAi approaches, pooled shRNA libraries have received considerable attention because of their versatility. A number of genome-wide shRNA libraries have been constructed against the human and mouse genomes, and these libraries can be readily applied to a variety of screens to interrogate the function of human and mouse genes in an unbiased fashion. We provide an introduction to the technical aspects of using pooled shRNA libraries for genetic screens.     

Cellular phenotyping by RNAi.

A systematic characterization of genes with unknown function is a key challenge after the sequencing of the human genome and the genomes of many model organisms. High-throughput RNA-interference (RNAi) screenings have become a widely used approach in invertebrate model organisms and also promise to revolutionize cell biology in mammals. Genome-wide RNAi screens in Caenorhabditis elegans and Drosophila, and in a smaller scale in mammalian cells have proven to be a valuable and successful method for the dissection of diverse biological processes. A number of RNAi libraries have become available that rely on different technologies, such as long double-stranded (ds) RNAs, in vitro diced short-interfering (si) RNAs, synthetic siRNAs and short-hairpin (sh) RNAs, which all have specific advantages and disadvantages. In addition, progress in screening technologies and data analysis allows the adaptation of screening methods to analyse more complex cellular processes. This review will summarize strategies in combining genome-scale RNAi libraries, high-throughput screening technologies, integrated high-content data analysis and will discuss future challenges.     

Biochemical mechanisms of the RNA-induced silencing complex

In less than 10 years since its inception, RNA interference （RNAi） has had extraordinary impact on biomedical science. RNAi has been demonstrated to influence numerous biological and disease pathways. Development and adoption of RNAi technologies have been prolific ranging from basic loss-of-function tools, genome-wide screening libraries to pharmaceutical target validation and therapeutic development. However, understanding of the molecular mechanisms of RNAi is far from complete. The purpose of this brief review is to highlight key achievements in elucidating the bio- chemical mechanisms of the RNA-induced silencing complex and to outline major challenges for the field.     

shRNA libraries and their use in cancer genetics

RNA interference was originally described as a powerful tool to inhibit gene expression in model organisms. Until recently, loss-of-function genetic screens in mammalian cells were hampered by a lack of suitable tools that can be used in a high-throughput format. Here we discuss the construction of short-hairpin RNA (shRNA) vector libraries, in particular those generated at the Netherlands Cancer Institute (NKI), and their application in mammalian cancer genetics. We describe their virtues and limitations, as well as different options for screening such libraries.     

Simultaneous analysis of large-scale RNAi screens for pathogen entry

Background

Large-scale RNAi screening has become an important technology for identifying genes involved in biological processes of interest. However, the quality of large-scale RNAi screening is often deteriorated by off-targets effects. In order to find statistically significant effector genes for pathogen entry, we systematically analyzed entry pathways in human host cells for eight pathogens using image-based kinome-wide siRNA screens with siRNAs from three vendors. We propose a Parallel Mixed Model (PMM) approach that simultaneously analyzes several non-identical screens performed with the same RNAi libraries.

Results

We show that PMM gains statistical power for hit detection due to parallel screening. PMM allows incorporating siRNA weights that can be assigned according to available information on RNAi quality. Moreover, PMM is able to estimate a sharedness score that can be used to focus follow-up efforts on generic or specific gene regulators. By fitting a PMM model to our data, we found several novel hit genes for most of the pathogens studied.

Conclusions

Our results show parallel RNAi screening can improve the results of individual screens. This is currently particularly interesting when large-scale parallel datasets are becoming more and more publicly available. Our comprehensive siRNA dataset provides a public, freely available resource for further statistical and biological analyses in the high-content, high-throughput siRNA screening field.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1162) contains supplementary material, which is available to authorized users.     

High-throughput RNA interference screening using pooled shRNA libraries and next generation sequencing

RNA interference (RNAi) screening is a state-of-the-art technology that enables the dissection of biological processes and disease-related phenotypes. The commercial availability of genome-wide, short hairpin RNA (shRNA) libraries has fueled interest in this area but the generation and analysis of these complex data remain a challenge. Here, we describe complete experimental protocols and novel open source computational methodologies, shALIGN and shRNAseq, that allow RNAi screens to be rapidly deconvoluted using next generation sequencing. Our computational pipeline offers efficient screen analysis and the flexibility and scalability to quickly incorporate future developments in shRNA library technology.     

Invertebrate models of spinal muscular atrophy: insights into mechanisms and potential therapeutics

Invertebrate genetic models with their tractable neuromuscular systems are effective vehicles for the study of human nerve and muscle disorders. This is exemplified by insights made into spinal muscular atrophy (SMA) using the fruit fly Drosophila melanogaster and the nematode worm Caenorhabditis elegans. For speed and economy, these invertebrates offer convenient, whole-organism platforms for genetic screening as well as RNA interference (RNAi) and chemical library screens, permitting the rapid testing of hypotheses related to disease mechanisms and the exploration of new therapeutic routes and drug candidates. Here, we discuss recent developments encompassing synaptic physiology, RNA processing, and screening of compound and genome-scale RNAi libraries, showcasing the importance of invertebrate SMA models.     

RNA interference in mammals: behind the screen

The discovery of RNA interference (RNAi) and the development of technologies exploiting its biology have enabled scientists to rapidly examine the consequences of depleting a particular gene product in a cell or an animal. The availability of genome-wide RNAi libraries targeting the mouse and human genomes has made it possible to carry out large scale, phenotype-based screens, which have yielded seminal information on diverse cellular processes ranging from virology to cancer biology. Today, several strategies are available to perform RNAi screens, each with their own technical and monetary considerations. Special care and budgeting must be taken into account during the design of these screens in order to obtain reliable results. In this review, we discuss a number of critical aspects to consider when planning an effective RNAi screening strategy, including selecting the right biological system, designing an appropriate selection scheme, optimizing technical aspects of the screen, and validating and verifying the hits. Similar to an artistic production, what happens behind the screen has a direct impact on its success.     

High-Throughput Screening by RNA Interference: Control of Two Distinct Types of Variance

The availability of genome-wide RNAi libraries has enabled researchers to rapidly assess the functions of thousands of genes; however the fact that these screens are run in living biological systems add complications above and beyond that normally seen in high-throughput screening (HTS). Specifically, error due to variance in both measurement and biology are large in such screens, leading to the conclusion that the majority of "hits" are expected to be false positives. Here, we outline basic guidelines for screen development that will help the researcher to control these forms of variance. By running a large number of positive and negative control genes, error of measurement can be accurately estimated and false negatives reduced. Likewise, by using a complex readout for the screen which is not easily mimicked by other biological pathways and phenomena, false positives can be minimized. By controlling variance in these ways, the researcher can maximize the utility of genome-wide RNAi screening.     

A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen

To enable arrayed or pooled loss-of-function screens in a wide range of mammalian cell types, including primary and nondividing cells, we are developing lentiviral short hairpin RNA (shRNA) libraries targeting the human and murine genomes. The libraries currently contain 104,000 vectors, targeting each of 22,000 human and mouse genes with multiple sequence-verified constructs. To test the utility of the library for arrayed screens, we developed a screen based on high-content imaging to identify genes required for mitotic progression in human cancer cells and applied it to an arrayed set of 5,000 unique shRNA-expressing lentiviruses that target 1,028 human genes. The screen identified several known and approximately 100 candidate regulators of mitotic progression and proliferation; the availability of multiple shRNAs targeting the same gene facilitated functional validation of putative hits. This work provides a widely applicable resource for loss-of-function screens, as well as a roadmap for its application to biological discovery.     

Enzymatically prepared RNAi libraries

Large-scale RNA interference (RNAi) screens in mammalian cells have mainly used synthetic small interfering RNA (siRNA) or short hairpin RNA (shRNA) libraries. The RNAi triggers for both of these approaches were designed with algorithm-based predictions to identify single sequences for mRNA knockdown. Alternatives to these approaches have recently been developed using enzymatic methods. Here we describe the concepts of enzymatically prepared shRNA and siRNA libraries, and discuss their strengths and limitations.     

Optimized PCR Conditions and Increased shRNA Fold Representation Improve Reproducibility of Pooled shRNA Screens

RNAi screening using pooled shRNA libraries is a valuable tool for identifying genetic regulators of biological processes. However, for a successful pooled shRNA screen, it is imperative to thoroughly optimize experimental conditions to obtain reproducible data. Here we performed viability screens with a library of ～10 000 shRNAs at two different fold representations (100- and 500-fold at transduction) and report the reproducibility of shRNA abundance changes between screening replicates determined by microarray and next generation sequencing analyses. We show that the technical reproducibility between PCR replicates from a pooled screen can be drastically improved by ensuring that PCR amplification steps are kept within the exponential phase and by using an amount of genomic DNA input in the reaction that maintains the average template copies per shRNA used during library transduction. Using these optimized PCR conditions, we then show that higher reproducibility of biological replicates is obtained by both microarray and next generation sequencing when screening with higher average shRNA fold representation. shRNAs that change abundance reproducibly in biological replicates (primary hits) are identified from screens performed with both 100- and 500-fold shRNA representation, however a higher percentage of primary hit overlap between screening replicates is obtained from 500-fold shRNA representation screens. While strong hits with larger changes in relative abundance were generally identified in both screens, hits with smaller changes were identified only in the screens performed with the higher shRNA fold representation at transduction.