"RNAi: Discovery to a functional map of the human genome"
siRNA -mediated RNA interference (RNAi) has become a key tool in functional genomics. In this presentation, we will discuss characteristic features of siRNAs which are critical for successful use of these reagents. We will first describe how combination of bioinformatics, chemical modifications and pooling strategy ensures high functionality of siRNAs, followed by an in depth discussion of one of the most critical issues in RNAi biology today- Specificity.
Specificity is critical, as non-specific or off-target effects may lead to false responses and misinterpretation of results. We will demonstrate how sequence alignment algorithms (e.g. Smith-Waterman) have limited ability to accurately filter siRNAs with potential off-target effects. Moreover, we will show that a primary source of off-target effects is derived from the “seed” region (nucleotides 2-8) of the siRNA’s antisense strand recognizing the 3’-UTR of an unintended target, resulting in silencing via a microRNA-like mechanism. We will then demonstrate that off-target effects can indeed cause false phenotypes. Finally, we will present strategies that effectively alleviate undesirable off-target effects and minimize the incidence of false phenotypes. The experimental approaches derived from these studies are expected to dramatically improve the quality and reliability of RNAi applications.
The acceptance of RNAi as a tool for functional genomics has led to an increasing number of gene silencing screens. These advances necessitate the development of reporting and data exchange guidelines that facilitate cross-laboratory data dissemination and analysis of large complex data sets. To that end, ten separate laboratories have simultaneously performed two consecutive screens based on cell viability and caspase activation using siRNAs targeting 859 related protein kinases and cell cycle regulators. Guidelines for MIARE Version 1.0 will be presented.
Lastly, an examination of the impact of a miRNA inhibitor screen on the onset and progression of hMSC to adipocytes in vitro will be presented.
"RNAi Global Initiative and high throughput screening
The RNAi Global Initiative represents an unprecedented venture bringing together a group of visionary research institutions and investigators to harness the potential of RNA interference as an important functional genomic tool.
Through collaboration and the meaningful exchange of information and data, we intend to draw a comprehensive roadmap of human gene function and use this as a foundation to revolutionize the way medicine and healthcare are delivered.
To this end, members of the RNAi Global Initiative are actively engaged in promoting the concept and implementation of minimum information standards to facilitate data sharing within the extended RNAi community. Building on established standards such as MIAME (Minimum Information About a Microarray Experiment), the RNAi Global Initiative has contributed work towards a community-wide effort known as the Minimum Information About an RNAi Experiment (MIARE). These reporting guidelines were
developed in part by a large inter-laboratory benchmarking study and in part by workshops and discussions amongst the RNAi Global Initiative members.
Members of the Genome-Wide RNAi Global Initiative
Charter Members of the Genome-Wide RNAi Global Initiative are listed below;
North America
British Columbia Cancer Research Centre Dharmacon, Inc.
Fox Chase Cancer Center Harvard Medical School
Stanford University School of Medicine
University of Nebraska Medical Center
• UNMC Eppley Cancer Center
University of Texas M.D. Anderson Cancer Center University of Texas Southwestern Medical Center
University of Toronto
• The Campbell Family Institute for Breast Cancer Research at Princess Margaret Hospital
• Samuel Lunenfeld Research Institute at Mount Sinai Hospital Yale University
Europe and Asia
Cancer Research UK (CRUK)
• London Research Institute
• Institute of Cancer Research
German Cancer Research Center (DKFZ) Institut Pasteur Korea (IP-Korea) Netherlands Cancer Institute (NKI)
University of Edinburgh Medical School
• Scottish Centre for Genomic Technology and Informatics (GTI)
University of Cambridge
• Cambridge Institute for Medical Research
• MRC Cancer Cell Unit
Vrije University Medical Center (VUMC) Weizmann Institute of Science (WIS)