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be exploited experimentally via the expression of synthetic short hairpin RNAs (shRNAs) to silence almost any gene10,11.
In other words, RNAi technology is a way for us to rapidly turn genes off and on. It serves as a fast alternative to gene deletion and, importantly, because it is reversible, gene silencing by RNAi bet- ter mimics the dynamics of small molecule inhibi- tion than permanent genetic knockouts. In a sense, it allows us to mimic drug therapy without the actual drug, which allows us to predict toxicities before we actually develop the drug. Since 1995, when the first pathways of RNAi were discovered, we have developed a much deeper understanding of how this process works enabling us to bring RNAi technology to its highest peak thus far. We have essentially learned how to hijack RNAi by delivering synthetic RNAs to effectively silence any gene of interest.
In the early days of RNAi, we utilised small syn- thetic interfering RNAs (siRNAs) that could be transiently delivered to cells12. Although rapid, cheap and effective for gene silencing, high concen- trations of siRNAs were often used, which had the potential to cause off-target effects and generate artifacts13-15. The second generation stem-loop short hairpin or shRNAs (so-named because of their structure) were a huge improvement because we could integrate them into the genome to get sta- ble gene suppression16. However, they too were not without flaws, often requiring screening of more than a dozen sequences to find an effective one, which discouraged some labs from using RNAi altogether.
Despite the skeptics, the field of RNAi has matured and found its way around this impedi- ment. We quickly realised that sequence really matters in using this technology. Rather than rely- ing on inaccurate prediction tools, our team, while at Cold Spring Harbor Laboratory, developed a high-throughput functional ‘sensor’ assay to unbi- asedly evaluate anywhere from 20,000 to 40,000 shRNA sequences in parallel17. We used this pro- cess to understand the requirements for effective RNAi and build a better system for potent gene knockdown18. We also learned that we were doing it wrong. By utilising only simple stem-loop shRNA structures, we were providing not one, but two RNA sub- strates because the passenger strand was also being incorporated into the RNA-induced silencing com- plex (RISC) at a high frequency, which can cause a lot of off-target effects and dilute the signal. One way to avoid this is to embed shRNAs into endoge- nous microRNA (miRNA) structures, guiding a
Drug Discovery World Fall 2017
Comprehensive tissue-based gene expression analysis services.
including detection of alternative splice variants and viral-vector expressed RNAs
• Any tissueall standard tissue preparations tissues, TMAs, frozen tissue and cell preps
• Tissue sourcingreliable sources for human and animal tissues
pathologist review • Quantitative image analysis HALO™ Software • 9,000+ catalog targets available • Two weeks for new probe design
• Four weeks turn-around time from receipt of samples to results for typical projects Extensive Experience
•10,000+ slides analyzed per year • 500+ assays validated and successfully performed
• 150+ tissue types including normal human, preclinical animals, clinical specimens, humanized mouse and syngeneic mouse tumor models
• Eight automated staining systems from Leica Biosystems and Ventana Medical Systems
Introducing BaseScope™ Assays
• Detect and visualize diverse RNA targets including splice variants, homologous sequences, CAR-T cell clones and more, all within the cellular morphological context
Learn more at
acdbio.com/pas
For Research Use Only. Not for diagnostic use. RNAscope is a registered trademark of Advanced Cell Diagnostics, Inc. in the United States or other countries. All rights reserved. ©2016 Advanced Cell Diagnostics, Inc.
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