RNA interference screening demystified The Breakthrough Breast Cancer Research Centre, The Institute of Cancer Research, London, UK Correspondence to: Dr C J Lord, The Breakthrough Breast Cancer Research Centre, The Institute of Cancer Research, Fulham Road, London SW3 6JB, UK; Chris.Lord@ icr.ac.uk Accepted 15 December 2008 Published Online First 6 January 2009 C J Lord, S A Martin, A Ashworth ABSTRACT Genetic screens, where the effects of modifying gene function on cell behaviour are assessed in a systematic fashion, have for some time provided useful information to those interested in disease pathogenesis and treatment. Genetic screens exploiting the phenomenon of RNA interference (RNAi) are now becoming commonplace. This article explains the different RNAi screen formats and describes some of the applications of RNAi screening that may be pertinent to the research pathologist. RNA INTERFERENCE (RNAI): WHAT, HOW AND WHY? Put simply, experimental RNAi allows the research scientist to assess the function of a gene or protein by silencing its expression using synthetic RNAs or plasmids (an effect often referred to as ‘‘knock- down’’). This technique exploits a physiological mechanism that represses gene expression by causing the degradation of protein-coding messen- ger RNA (mRNA) transcripts. In mammalian cells, physiological RNAi is primarily mediated by non- protein-coding RNA transcripts, known as microRNAs (miRNAs) (fig 1). miRNAs are pro- duced in much the same way as normal mRNAs, but instead of being translated into proteins, miRNAs are processed into shorter RNA species containing a hairpin structure, known as short- hairpin RNAs (shRNAs). These are in turn processed into short double-stranded pieces of RNA known as short-interfering RNAs (siRNAs). Within a multiprotein complex known as RISC (RNA-induced silencing complex), one strand of a siRNA duplex binds a protein-coding mRNA transcript that bears a complementary nucleotide sequence. This interaction allows a nuclease in RISC to cleave and destroy the protein-coding mRNA, thus silencing the expression of the gene in a relatively sequence-specific manner (fig 1). In 1998, Mello and Fire demonstrated that potent gene silencing could be experimentally induced by injecting double-stranded (ds)RNA into the nematode Caenorhabditis elegans. In this con- text, dsRNA is processed into siRNA by an enzyme, Dicer.1 In mammalian cells, however, the introduction of long dsRNAs induces an interferon response that confounds experimental results, primarily by inducing an interferon response that causes a global shutdown of protein synthesis.2 However, synthetic siRNAs, or plas- mids expressing shRNAs or miRNAs which are processed into siRNAs, can be introduced into mammalian cells and mediate gene silencing with- out significant adverse effects.2 The experimental use of synthetic siRNAs and shRNA plasmids has profoundly changed the way This paper is freely available online under the BMJ Journals unlocked scheme, see http:// jcp.bmj.com/info/unlocked.dtl J Clin Pathol 2009;62:195–200. doi:10.1136/jcp.2008.058735 Demystified in which the loss of function of particular genes or proteins is studied. Previously, techniques that were either more time consuming, such as gene targeting (making ‘‘knockout’’ cell lines or ani- mals), or capricious, such as antisense RNA, were used. Alternatively specific inhibitors of a particu- lar protein could be used, but these are limited in scope. Now RNAi reagents can be purchased and trivially used to silence almost any gene in the genome. As described above, the types of RNAi reagents fall into two main categories, siRNAs and shRNA expression constructs. Table 1 shows the advan- tages of each reagent format. siRNAs consist of small double-stranded pieces of RNAs (generally over 19 bp), one strand of which matches a complementary sequence in the gene to be knocked down. These siRNAs can be either synthetically synthesised or generated by enzyme digestion of mRNA, a technique known as esiRNA.3 The main advantage of siRNAs is ease of use; given their small size, siRNAs are readily transfected into most cell types, and they can be purchased in ‘‘ready-to- transfect’’ format from many commercial suppli- ers. One limitation of siRNAs, however, is that gene silencing is only achieved over a short period of time; siRNAs are most likely degraded by the host cell and are also not replicated along with the host cell’s DNA. Therefore, as cells prolifer- ate, the proportion of cells carrying siRNA is gradually reduced. To some extent, this short period of silencing (a number of days) has been addressed by chemical modification of siRNA (see www.dharmacon.com), but if long-term silencing is required, shRNA expression constructs are more often the solution. These constructs express miRNAs or shRNAs that are processed by the target cell’s own RNA-processing machinery into siRNA, which in turn mediate gene silencing. These constructs come in a variety of formats and can be purchased either as simple plasmids or as viral constructs that are easier to deliver to a wider range of cell lines. shRNA vector-based reagents can provide long-term, stable gene silencing, as the vectors used are able to integrate into genomic DNA and are thus copied along with cellular DNA. Although the inherent instability of some of the initial viral systems used to express shRNA limited their utility, this problem has now been addressed in the most recent versions that are available from commer- cial suppliers. One of the more recent advances in shRNA construct design has been the use of vectors that express miRNAs instead of shRNAs. It is proposed that, as this process more closely mirrors the mechanism by which endogenous mammalian miRNAs mediate gene silencing, it leads to an improvement in silencing efficiency.4 5 195