New Possibilities arise in RNAi Screening with the Introduction of the Nucleofector 96-well Shuttle System
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The recent explosion of interest in applications such as screening siRNA libraries for target identification and validation requires the ability to quickly process large numbers of samples. While lipofection suffices to perform siRNA screening experiments in easy-to-transfect cell lines such as CHO or COS cells, transfection of demanding-to-transfect cell types such as cancer cell lines, suspension cell lines or primary cells, which are often the favored biological set-up, remains a challenge.
To address these needs the Nucleofector technology has now been expanded to a 96-well format. The new Nucleofector 96-well Shuttle System provides researchers with the possibility of integrating the well established Nucleofector technology into high throughput processes. And because the 96-well Shuttle System is based on the Nucleofector technology, the optimal transfection protocols remain identical for a specific cell type, whether you are transfecting DNA or RNA, which can be particularly advantageous for establishing RNAi experiments.
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RNAi
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Gene silencing by RNA interference (RNAi) is one of the biggest breakthroughs in the past decade, rapidly emerging as a powerful technology for assessing gene function in mammalian cells, and becoming a valuable tool in functional genomics, target discovery and validation.
RNAi mediated gene silencing relies on small (typically 21-23 bp) double-stranded RNA molecules which must be identical in sequence to the cognate gene. The presence of such small inhibitory RNA (siRNA) molecules leads to specific cleavage and destruction of the mRNA target by cellular enzymes in an evolutionarily conserved and ubiquitous process.
Recently there has also been increased interest in a related species, microRNAs (miRNA), which are encoded in the genomes of plants, animals and their viruses. These highly conserved ~21-mer RNAs appear to regulate gene expression by binding to the 3'-untranslated regions of specific mRNAs and inhibiting translation. Exact sequence identity with the target sequence is not essential and so many miRNAs are thought to regulate multiple genes. With hundreds of miRNAs predicted to exist in higher eukaryotes, the potential regulatory circuitry afforded by miRNA is considerable. A collection of miRNA sequences from numerous species has been compiled in the online miRBASE (http://microrna.sanger.ac.uk/sequences/index.shtml).
For experimental purposes siRNA (or miRNA) duplexes are almost exclusively of either synthetic origin or expressed from plasmids within cells (usually as short hairpin (sh)RNA). Chemically synthesized duplexes offer a fast way to find the siRNA sequence that results in the most effective knockdown. However, working with shRNA expressed from plasmids is often preferred as DNA is more stable (easier to grow, handle and store) and allows for the generation of stable RNAi-expressing cell lines. Using a technology which permits switching between substrates in cell lines and primary cells without having to re-optimize transfection conditions, offers a considerable savings in both time and effort.
While it is an extremely powerful approach, RNAi experiments must be carefully designed and must include appropriate controls in order to be assured that the experimental results arise from the specific silencing of a particular gene product. These concerns were addressed in a 2003 Nature Cell Biology editorial (1) which suggested that the control of choice for RNAi experiments should be rescue of the biological effect by expression of the target gene in a form refractory to siRNA. In practical terms this means co-transfecting cells with siRNA directed against 3'untranslated region of the target gene together with a plasmid expressing its cDNA. A more involved alternative (appropriate for siRNAs targeting the coding region of mRNA transcripts) is to co-transfect the siRNA with a plasmid expressing a copy of the target gene containing one or more silent third-codon point mutations within the targeted region. The ability to transfect DNA and RNA using identical conditions means that these types of experiments are quite easy to perform using the Nucleofector technology.
Algorithms for design of siRNA duplexes continue to improve, however, it is still essential to test multiple sequences in order to be certain of finding one that is both highly effective and yet absolutely specific in down-regulating the target gene. Furthermore, in order to minimize the possibility of non-specific effects (2), it is important to keep siRNA concentrations as low as possible and ideally results should be confirmed with two or three separate siRNAs directed against the same target (1) so even simple RNAi experiments can require a significant number of samples just to establish the optimal experimental conditions. And this gets correspondingly more complicated when large numbers of genes must be examined, such as when screening RNAi libraries.
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RNAi libraries
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In the search for novel drug targets and analysis of signaling pathways it is often important to analyze a whole set of target genes (which may belong to the same pathway, be part of a specific class of proteins or cover the entire genome). Such analyses can be performed using RNAi libraries based on either RNA duplexes or DNA plasmids.
Each of these methods offers particular advantages. A large number of libraries based on synthetic duplexes are readily available from most major siRNA suppliers, and range from individual signaling pathways to the entire genome. As these commercially available siRNA libraries do not require any hands-on-time from the researcher, are often subject to certified production processes and assure efficient knockdown with validated sequences, they are usually the substrate of choice with which to start screening experiments. In addition, chemically synthesized siRNA duplexes can be labeled with fluorescent tags, which allow an easy means of visualizing transfection efficiency.
Libraries of shRNA-expressing vectors are traditionally more difficult to develop, but these vectors have advantages in terms of general handling (DNA is more stable and hence easier to handle and store) and propagation (for large-scale approaches, growing plasmids can be more cost-effective than chemical synthesis of siRNA). Furthermore, having the shRNA expression cassettes in plasmids makes it easier to subsequently generate stable RNAi-expressing clones, to include reporter or antibiotic markers (for selection of transfectants), and/or to incorporate inducible promoters to allow the knockdown phenotype to be switched on and off.
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Primary cells
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An important consideration for screening RNAi libraries is the type of cell used. Primary cells, directly isolated from living tissue, lack the cellular transformations that permit the unlimited growth of cell lines. This makes them difficult to maintain in culture for prolonged periods of time but also means that they more closely represent the cells as they exist within the organism. This is of particular importance for drug development: the more predictive the first screens are, the easier it is to determine which targets or compounds to focus on for further development. This can save considerable amounts of both time and money.
As the ultimate goal of most research is to gain information directly applicable to the living organism, it is ideal to use a model as close as possible to the in-vivo situation. Thus, for most purposes, primary T cells provide a superior model for immunological research than, for example, the T cell line Jurkat, and primary neurons are a better model for neurological research than the commonly used cell line PC-12.
Ideally, primary cells would be used in every phase of the discovery process – from target identification and validation to pre-clinical studies. In the field of initial compound screens the share of cell-based assays has grown from only a few percent some years ago to approximately 50% at present, and it can be safely assumed that the proportion of cell-based assays performed with primary cells will continue to grow in the future.
In contrast to other non-viral transfection methods, such as lipofection or conventional electroporation, the Nucleofector technology allows DNA to directly enter the cell nucleus (rather than having to wait for breakdown of the nuclear membrane during cell division as is the case for other non-viral transfection systems). It is because of this nuclear transport that slowly and non-dividing primary cells (such as neurons) can be so efficiently transfected by nucleofection. It also means that expression of transfected genes begins almost immediately, significantly reducing the time of analysis for most experiments.
The Nucleofector technology is equally well suited for siRNA duplex delivery and adds many hard-to-transfect cell lines to the spectrum of cell lines that are now amenable to such studies. Moreover, nucleofection also enables co-transfections of multiple (different) substrates, allowing for co-expression of genes from multiple plasmids, or co-transfection of plasmids in combination with siRNA duplexes.
With the expansion of the Nucleofector technology to a 96-well format, researchers can now fully exploit both of the two major trends for screening RNAi libraries: the trend to switch between transfecting siRNA duplexes and shRNA-expressing plasmid vectors, and the trend towards using the biologically most relevant model system, i.e. primary cells.
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