Demystified Figure 1 RNA interference. For simplicity we have shown how microRNAs (miRNAs) can mediate RNA interference in mammalian cells by causing the degradation of protein-coding transcripts. Non-protein coding miRNAs are transcribed from the genome and then processed in the nucleus into shorter RNA species bearing a hairpin structure (shRNAs); these hairpin structures, consisting of a ‘‘stem’’ and a ‘‘loop’’, are caused 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’’). shRNAs are exported from the nucleus and further processed into small RNA duplexes (siRNAs) formed from the stem of the shRNA. siRNAs are loaded into the RNA-induced silencing complex (RISC). This complex facilitates binding between one of the siRNA strands and protein-coding mRNAs that have nucleotide sequence complementary to the siRNA. Once siRNA/mRNA binding has occurred, and thus the target mRNA transcript has been recognised, a nuclease in RISC degrades the mRNA, thus ultimately reducing the amount of mRNA that is available for translation and protein production. This mechanism can be exploited experimentally to silence specific genes. Synthetic siRNAs can be delivered into cells by transfection and are readily loaded on to the RISC and mediate degradation of mRNAs with significant sequence complementarity. Viruses or plasmids containing miRNA-coding or shRNA-coding sequences can also be introduced into mammalian cells and these mimic the production of endogenous miRNA and shRNA and are processed into siRNA as before. It is also worth noting that these improvements in vector design also raise the possibility of in vivo RNAi screens in whole animals, as opposed to in vitro screens in cultured cells. Already, individual genes can be targeted in mice using RNAi,6 and, in fact, candidate tumour suppressor genes have been validated using this approach.7 siRNAs and shRNA expression constructs are now available as libraries that contain reagents targeting any number of genes. Depending on the application, RNAi reagents targeting a single gene, a particular gene subset, for example protein kinases, or indeed the entire genome may be purchased. The commercial availability of such reagents now means that a number of genes can be screened simultaneously, and relatively straightfor- wardly, for their involvement in a chosen biological phenotype. The format of how such a screen is performed is largely determined by the choice of RNAi reagent. Both shRNAs and siRNAs are well suited to ‘‘one gene per well’’ screens (fig 2) where cells are transfected or infected in multi-well plates, each well containing RNAi reagents that silence one gene. Normally 196 96-well or 384-well plates are used for such screens, meaning that the entire genome can be screened using under two hundred 384-well plates or just over six hundred 96-well plates. Obviously such screens are better performed using some form of liquid handling automation, although smaller screens where a gene subset such as the protein kinases are targeted can be performed manually using only ten 96-well plates. The simplest screens of this type use some form of plate reader to measure the effect of RNAi reagents; measuring cell viability by the use of an ATP colorimetric assay is common. However, the recent trend is to measure multiple phenotypes within the same screen, sometimes using high-throughput microscopy, an approach termed ‘‘high-content’’ analysis. For example, Bakal et al8 recently screened a small gene subset by RNAi using high- throughput microscopy to measure changes in cell morphology. Although the computational requirements to analyse such high- content screens properly are high, these approaches offer the potential to dissect complex biological networks and pathways. The scale of whole-genome screens that involve the use of hundreds of multi-well plates can often be daunting. Given this, an alternative approach is to use shRNA-expression constructs in ‘‘pooled’’ screens (fig 2). Here, pools of shRNA-expressing vectors are used to infect or transfect cells, which are then selected for the phenotype of interest such as resistance to a small-molecule inhibitor. The target gene in surviving cells is identified by PCR amplification and sequencing of the unique short hairpin sequence of the shRNA vector that encodes the gene-specific shRNA. Relatively new sequencing platforms, such as ABI SOLiD (Roche 454) and Illumina’s Genome Analyzer II are ideal for this purpose as they are able to simultaneously sequence millions of short pieces of DNA, such as PCR products containing short hairpin sequences of shRNAs. An alternative approach for screening deconvolution is to use oligonucleotide microarrays to identify hits from pooled shRNA screens. These technologies can be used to identify shRNA targets that are either selected for (positive selection) or selected against (negative selection). For example, Brummelkamp and colleagues9 used a library targeting ,8000 genes in a positive bar code screen to identify mediators of sensitivity to the anticancer drug Nutlin-3, an inhibitor of Mdm2 that activates the p53 pathway, allowing a more complete understanding of the mechanism of tumour selectivity for this drug.9 Such pooled approaches are not limited to drug-sensitisation screens. For example, Green and colleagues screened ,62 400 shRNAs targeting ,28 000 mouse genes to analyse the mechanisms that control the cell surface expression of the pro-apoptotic Fas receptor.10 The ,62 400 shRNAs were divided into 10 pools, which were packaged into retrovirus particles and used to stably transduce Fas receptor-negative, K-ras NIH 3T3 cells. Cells in which Fas receptor expression had been induced by RNAi were selected from the total cell population using immunomagnetic beads bearing an anti-Fas antibody. This Fas receptor-positive population was expanded, and the shRNAs that caused expression of the receptor were identified by sequencing shRNAs present in a FasR-positive population. Ultimately this screen identified a mechanism by which the oncogene Ras mediated epigenetic silencing of the Fas gene, thus allowing tumour cells to avoid apoptosis, one of the hallmarks of a cancer cell. THE PROCESS The commercial availability and relative ease of use of RNAi libraries now means that most research labs are able to perform an RNAi screen, be it genome wide or targeting a smaller gene J Clin Pathol 2009;62:195–200. doi:10.1136/jcp.2008.058735