Glossary c RNAi, RNA interference. The process by which RNAs that encode proteins (messenger RNAs (mRNAs)) are degraded in a sequence-specific manner by binding to a short interferring RNA. c siRNA, short interfering RNA. The RNA species that binds mRNA in the RNA-induced silencing complex (RISC). The mRNA/siRNA interaction activates the nuclease activity of RISC, leading to degradation of the mRNA. c shRNA, short hairpin RNA. A siRNA precursor, encompassing a ‘‘stem’’ and a ‘‘loop’’ structure formed by base pairing between short regions of the RNA sequence (these form the ‘‘stem’’) separated by a short sequence that does not form base pairs (which forms the ‘‘loop’’). c microRNA (miRNA), a non-protein-coding RNA species. One of the known functions of miRNAs in mammalian cells is in RNA interference c pooled/non-pooled screens. In non-pooled screens, RNAi reagents targeting one gene are introduced into a single-cell population. Non-pooled screens are normally carried out in multi-well plates, with each well representing RNAi reagents targeting one gene. In pooled screens, RNAi reagents targeting a number of genes are introduced into the cell population at the same time. c Infection, the process by which DNA, packed within a viral coat, is introduced into cells. DNA-encoding miRNAs or shRNAs can be packaged as virus and infected into cells. c Transfection, the process by which ‘‘naked’’ or non-virally packaged DNA is introduced into cells. Normally this is achieved by binding DNA to a lipid; the resultant lipid sphere binds to and merges with the target cell’s plasma membrane, releasing the DNA into cells. screen. Subsequent to this technical validation, it is necessary to eliminate the possibility of off-target effects, ie, effects that are due to silencing of genes other than the intended targets. Although silencing of genes via RNAi requires high comple- mentarity to the target mRNA, off-target effects may require considerably less, in a similar fashion to the microRNA machinery. A 7–8-nucleotide ‘‘seed’’ region at the 59 end of the anti-sense strand is most critical in generating off-target effects, particularly when this seed region occurs in the 39 untranslated region of a potential off-target gene.16 At present it is not possible to completely eliminate off-target effects by reagent design alone, although some of the commercial suppliers now sell siRNAs that are chemically modified to limit such effects. However, although some of these chemical modifica- tions are able to reduce off-target effects, they may compromise silencing efficiency. Ultimately, to demonstrate that knock- down of the targeted gene is directly responsible for the phenotype, it is important to perform ‘‘rescue or redundancy’’ experiments.17 Rescue may involve reversing the phenotype induced by the siRNA by re-expressing an siRNA-resistant cDNA (one that has mismatches with the siRNA and cannot be targeted for degradation). With the large number of hits that result from RNAi screens, this is often not possible, and, in many cases, overexpression of a cDNA has aberrant effects that make this approach difficult to implement. In most cases, redundancy is sufficient to confirm specificity. This involves demonstrating that multiple RNAi reagents targeting different regions of the same target mRNA cause the same phenotype. J Clin Pathol 2009;62:195–200. doi:10.1136/jcp.2008.058735 Demystified Take-home messages c RNAi screens are now becoming part of everyday technology used in the research laboratory. c There are a number of ways to use this technology based on the type of question asked and the scale of the undertaking. c RNAi technology has the potential to dissect basic biological phenotypes, as well as providing insight into clinically relevant questions. Silencing caused by individual RNAi reagents should also be confirmed by quantitative real-time PCR or western blotting the target protein. As alternative approaches, small-molecule inhibitors of the ‘‘hit’’ proteins may be used to show that loss of function can elicit the particular phenotype, as can the use of ‘‘dominant-negative’’ cDNA expression constructs or inhibiting antibodies. Validating individual hits from a screen can often be a more time-consuming and expensive enterprise than the screen itself. Given this, one may choose to use a more rapid approach that exploits the improvements in bioinformatic analysis that are now available. For example, one can now take the list of ‘‘hit’’ genes from a RNAi screen and subject this list to some form of pathway analysis; where a number of hits from the screen fall into the same biological pathway, or indeed have a well- established physical or function interaction, one has some confidence that the effects are real. To achieve this, one has the option of using many of the pathway analysis tools that are publicly available or even the commercial tools such as Ingenuity (www.ingenuity.com). However, it must be noted that this approach is only as good as the quality of the data in the pathway or interaction database that is used. Furthermore, this approach to validation is perhaps only appropriate for whole-genome RNAi screens, as particular gene subset screens may already be biased towards particular pathways. APPLICATIONS FOR THE PATHOLOGIST As the reader will see below, RNAi screens represent more of a research tool rather than an application that could be used in the diagnostic laboratory. Nevertheless, individual RNAi reagents, rather than RNAi libraries and screens, do serve one obvious application for use in the diagnostic laboratory. The ability of RNAi reagents to knockdown the expression of specific proteins makes them ideal for the validation of antibodies that are to be used in diagnostic tests. For example, cells transfected with siRNAs targeting one gene/protein, when compared with control-transfected cells, serve as ideal controls for identifying the optimal conditions for the use of an antibody. Where RNAi screens are involved, the interest to the pathologist is their utility in the dissection of basic cellular pathways, but also in more translational areas such as identifying novel therapeutic approaches and mechanisms of drug resistance. To illustrate this, we present a number of ‘‘case reports’’ describing a selection of the more pertinent RNAi screens. Case history 1: viral infection Flaviviruses, such as West Nile virus, represent a significant risk to human health. To understand what determines whether a cell can be infected with West Nile virus, Krishnan and 199