Demystified Table 1 RNAi library formats Library type Advantages siRNA c Consistent quality of reagents c normally arrayed in 96-well or 384- c Ease of use and readily transfectable well plates c Chemical modification of siRNA can c can combine multiple siRNAs limit off-target effects targeting different sequences in the same gene in one well (SMARTPools) c can be purchased in ‘‘ready-to- transfect’’ aliquots or in larger amounts that require replating c can also be arrayed on slides shRNA as: c Renewable resource c plasmid DNA arrayed in 96-well or c Viral vectors enable silencing in 384-well plates for transfection difficult-to-transfect cells c viral particles in multi-well plates c Suited to pooled screens as well as (one shRNA per well) arrayed screens c pools of plasmids or viral particles c Stable integration of silencing machinery into host cell genome enables longer-term silencing c Varying vector formats available that allow inducible silencing, tracking of silencing machinery, etc Disadvantages c Finite resource c Target cells need to be transfectable c Relatively short period of silencing c Not suited to pooling strategies c Reduced ease of use: require preparation of plasmid DNA and, in the case of viral-based libraries, viral packaging c Viral use often requires biological containment c Pooled screens require significant deconvolution Supplier websites: www.openbiosystems.com; www.dharmacon.com; www.origene.com; www.scbt.com; www.ambion.com; www.sigmaaldrich.com; www.systembio.com. subset. Given this, it is worth describing the major issues that should be considered before embarking upon such an enterprise. As with any well thought out experiment, the screen assay to be measured must carefully reflect the clinical, pharmacological or biological question. The assay system must also be robust, reproducible and affordable. Most laboratories use human tumour cell lines for RNAi screens, as these can be routinely grown and transfected but, depending on the phenotype examined, the use of a non-human model system might be considered. The biggest advantage is cost, which can be significantly lower in model organisms because of the ability of C elegans and Drosophila melanogaster to take up long dsRNAs by feeding or soaking methods, eliminating the requirement for expensive transfection reagents. Recent studies indicate that many results from RNAi screens in model organisms translate to humans.11 Of equal importance is the choice of RNAi library. Given the many different formats of RNAi libraries available, this decision is governed by the choice of a pooling or non- pooled approach. If a pooling strategy is chosen, screens have been mostly limited to the vector-based libraries. Whether the assay system requires long-term or short-term gene silencing also needs to be taken into account. Stable integration of vector backbones combined with antibiotic selection cassettes make vector libraries advantageous if long-term silencing is a necessity. However, non-vector siRNA libraries should not be discounted, as transfection efficiencies are normally higher using these smaller nucleic acids, and significant gene silencing can often still be observed after 7 days. Finally, there is the question of reagent lifespan. Vector-based libraries are poten- tially renewable, but this can be time consuming and expensive. Many prefer the option of having a ‘‘ready-to-go’’ reagent, acknowledging that silencing technology is still improving. Finally, as with any experiment, positive and negative controls should ideally be used in RNAi screens. These controls allow the dynamic range of the assay to be estimated and also, if replicated several times within a library, serve as a form of data quality control. Ideally a negative control would take the form of an RNAi reagent that engages the gene-silencing apparatus but silences a gene that has no effect on the phenotype of J Clin Pathol 2009;62:195–200. doi:10.1136/jcp.2008.058735 interest, such as a synthetic gene in the target cell line such as GFP, the silencing of which can be readily monitored. For example, using cell lines that express a fluorescent protein, such as GFP, in combination with an siRNA that targets GFP, would provide an ideal negative control for many screens. However, the use of such a system may restrict the user to a limited number of cell lines, and the norm is to use non-targeting siRNA.12 Although some commercial non-targeting controls are well suited to this purpose, some induce off-target effects (see below) that lead to cell death, and the user is well advised to assess the effect of each control on the phenotype in question, comparing with mock-transfected cells. Positive controls can be used to define the magnitude of change that is thought to constitute a meaningful biological effect and verify that the screen is returning valid biological data. They also provide biologically relevant thresholds for identifying positive hits from a screen. Comparison of positive and negative controls also allows the screening window coefficient or Z factor13 to be estimated, which describes the suitability of the assay system for large-scale screening. Checking that the screen is suitably robust reduces statistical false positives and negatives and saves time and money spent on wasted screen data or attempting to validate false positives. Once decisions have been made on the factors described above, a typical screening strategy is as follows. Firstly, experimental parameters need to be optimised. Probably, the best place to start is to ensure that high-efficiency transfection or infection can be achieved in the format (multi-well plate or other) that is to be used for the screen. Ideally, one is looking for transfection/infection conditions that enable efficient delivery of the RNAi reagent without causing excessive, non-specific toxicity. One simple trick for assessing RNAi delivery para- meters is to transfect or infect cells with RNAi reagents that silence genes that are essential for cell viability and compare cell death with control transfected cells. In human tumour cells, RNAi reagents targeting Polo-like kinase 1 are often used for this purpose, as knocking down this kinase consistently kills most cancer cells.14 Different transfection reagents and procedures should be tried here, but more often than not the best place to 197