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| 190 | OLD Functional Genomics Lab Expertise | Historically, a lack of methods to properly interpret the results of genome-wide screens, a lack of collaborative expertise to perform RNAi experiments, and the absence of comprehensive RNAi data in public databases for researchers to reference all have limited RNAi’s usefulness. To address these problems, NCATS operates a state-of-the-art RNAi and CRISPR screening facility open to NIH investigators.The Functional Genomics Lab experts routinely perform common seed analysis, multiple siRNA testing and separation of false positives from true positives and are extending this platform to develop similar methodologies for CRISPR/Cas9-based screening.Functional Genomics Lab ExpertiseCommon Seed Analysis to Prioritize Hits and On- or Off-Target EffectsA common source of error in RNAi experiments is when RNAi turns off genes other than the intended targets. These “off-target effects” can be detected by comparing the experimental results of an RNAi to all other similar RNAi, enabling scientists to determine if the effect observed is specific to the gene being targeted or if it occurs with all similar RNAi.In this plot, red triangles indicate the experimental results from siRNAs that turn off the SIAH3 gene. Black circles are plotted for all similar RNAi and demonstrate clearly that the results for SIAH3 are separated from these and are not an error caused by off-target effects.Testing Multiple siRNAs to Increase Confidence in Genes with ConsensusBecause many genes are identified in error in a typical RNAi screen, follow-up experiments using different RNAi are an important way that scientists can gain confidence in their results.In this bar plot, the number of active siRNAs identified for each gene is displayed for the primary (initial screen) and follow-up experiments.Separation of False Positives from True Positives Using C911 ControlsIn addition to common seed analysis, RNAi experts at NCATS have developed a novel technology to validate identified genes: C911 controls. C911 siRNAs are modified siRNAs that retain off-target effects but eliminate the normal function of the siRNA.By comparing experiments using an siRNA to experiments using the same siRNA with the C911 modification, scientists can tell the difference between true (green plot) and false (red plot) positive results.Functional Genomics Lab ResourcesWe employ scientific resources that include a variety of commercially available siRNA screening libraries; tools for data tracking, storage and analysis; and a fully automated, large-scale screening platform.Screening LibrariesAmbion Silencer Select Human Genome-Wide siRNA library targeting ~22,000 genes with three individual siRNAs per geneAmbion Silencer Mouse Genome-Wide siRNA library targeting ~17,000 genes with three individual siRNAs per geneDharmacon siGENOME and Dharmacon ON-TARGETplus Human Genome-Wide siRNA libraries (Thermo Scientific), with pools of four siRNAs targeted against each of ~16,000 genes in the library; “druggable genome” subsets included for focused screening of potentially druggable targetsmicroRNA mimic and inhibitor librariesAmbion Silencer Select Human Druggable Genome siRNA Library V4Dharmacon Human ON-TARGETplus siRNA Transcription Factors LibraryDharmacon Human ON-TARGETplus Epigenetics siRNA LibraryAmbion Silencer Select Human Ubiquitin 96 siRNA LibrarySoftware and Analysis ToolsRNAi Data Viewer This tool allows users to view plates, check stats, screen data and perform a variety of analyses. Access user data (restricted).AssaysSimple phenotypesPrimary reporter assays Complex phenotypesRobotic PlatformLiquid handlers, washers and dispensersEnVision and Pharastar Multilabel Reader (PerkinElmer)Measures luminescence, fluorescence, fluorescence polarization, absorbance and time-resolved fluorescenceImageXpress Micro XL confocal high-content imager (Molecular Devices)Automated microscope used for the acquisition and analysis of cell-based images in relatively high throughput | ||||||||
| 177 | Functional Genomics Lab Scientific Capabilities | Historically, a lack of methods to properly interpret the results of genome-wide screens, a lack of collaborative expertise to perform RNAi experiments, and the absence of comprehensive RNAi data in public databases for researchers to reference all have limited RNAi’s usefulness. To address these problems, NCATS operates a state-of-the-art RNAi and CRISPR screening facility open to NIH investigators. NCATS staff assist NIH investigators with all stages of project planning and execution, from assay development through genome-wide siRNA screens, informatics and pathway analysis, and rigorous confirmation of results. The Functional Genomics Lab experts routinely perform common seed analysis, multiple siRNA testing and separation of false positives from true positives and are extending this platform to develop similar methodologies for CRISPR/Cas9-based screening. Learn more about the Functional Genomics Lab’s available resources and expertise. | ||||||||
| 176 | Functional Genomics Lab in Action | The Functional Genomics Lab has initiated a systematic evaluation and comparison of RNAi and CRISPR/Cas9-based gene-editing technologies. The objectives of this evaluation are to understand the utility and pitfalls of these newer technologies and to establish the experimental, automation and computational workflows required for focused, arrayed, small-scale CRISPR-based screens of approximately 400 to 600 genes. This work was initiated in June 2017.The Functional Genomics Lab’s evaluation plan has three components:Reagent selectionRobust screening workflow developmentAssessment of the relative strengths of each functional genomic approach to ensure the selection of the most appropriate technologies needed to address the specific question under investigationSeptember 2017TNRF Receives Deputy Director of Intramural Research (DDIR) Innovation Award (PDF - 604KB) TNRF received a collaborative DDIR Innovation Award two years in a row, both in May 2017 and May 2018, to develop proof-of-concept pilot CRISPR screens (arrayed and population-based) that bring in new investigator-driven expertise to develop complementary CRISPR-based approaches. These pilot screens are a mix of hypothesis-generating and hypothesis-driven studies to fully test different aspects of single-guide RNA-CRISPR-based screening platforms.July 2017RNAi High-Throughput Screening of Single- and Multi-Cell-Type Tumor Spheroids: A Comprehensive Analysis in Two and Three Dimensions The widespread use of two-dimensional (2-D) monolayer cultures for high-throughput screening (HTS) to identify targets in drug discovery has led to attrition in the number of drug targets being validated. Solid tumors are complex, aberrantly growing microenvironments that harness structural components from stroma, nutrients fed through vasculature and immunosuppressive factors. Increasing evidence of stromally derived signaling broadens the complexity of our understanding of the tumor microenvironment while stressing the importance of developing better models that reflect these interactions. Three-dimensional (3-D) models may be more sensitive to certain gene-silencing events than 2-D models because of their components of hypoxia, nutrient gradients and increased dependence on cell-cell interactions and therefore are more representative of in vivo interactions.June 2017Exploring Drug Dosing Regimens In Vitro Using Real-Time 3-D Spheroid Tumor Growth Assays Two-dimensional monolayer cell proliferation assays for cancer drug discovery have made the implementation of large-scale screens feasible but seem to reflect only a simplified view that oncogenes or tumor suppressor genes are the genetic drivers of cancer cell proliferation. However, there is now increased evidence that the cellular and physiological context in which these oncogenic events occur play a key role in how they drive tumor growth in vivo and, therefore, in how tumors respond to drug treatments. In vitro 3-D spheroid tumor models are being developed to better mimic the physiology of tumors in vivo, in an attempt to improve the predictability and efficiency of drug discovery for the treatment of cancer. Here we describe the establishment of a real-time 3-D spheroid growth, 384-well screening assay. The cells used in this study constitutively expressed green fluorescent protein (GFP), which enabled the real-time monitoring of spheroid formation and the effect of chemotherapeutic agents on spheroid size at different time points of sphere growth and drug treatment. This real-time 3-D spheroid assay platform represents a first step toward the replication in vitro of drug dosing regimens being investigated in vivo. We hope that further development of this assay platform will allow the investigation of drug dosing regimens, efficacy and resistance before preclinical and clinical studies.December 2013Gene-Silencing Data Now Publicly Available to Help Scientists Better Understand Disease On Dec. 11, 2013, NIH announced that for the first time, large-scale information on the biochemical makeup of small interfering RNA (siRNA) molecules is available publicly. NCATS researchers collaborated with Life Technologies Corporation of Carlsbad, California, which owns the siRNA information, to make it available to all researchers.November 2013Gene-Silencing Study Finds New Targets for Parkinson’s Disease On Nov. 25, 2013, NIH announced that NCATS and National Institute of Neurological Disorders and Stroke researchers had used RNAi technology to identify dozens of genes that may represent new therapeutic targets for treating Parkinson’s disease. The research was published online in Nature(link is external).March 2013Functional Genomic Screening Identifies Dual Leucine Zipper Kinase as a Key Mediator of Retinal Ganglion Cell Death On March 5, 2013, NCATS scientists published results of an RNAi screening study identifying a protein that could represent a therapeutic target for glaucoma.February 2013Human Genome-Wide RNAi Screen Reveals a Role for Nuclear Pore Proteins in Poxvirus Morphogenesis An article published on Feb. 26, 2013, revealed that a human genome-wide RNAi screen had identified candidate genes that modulate the activity of vaccinia virus, representing possible therapeutic targets. | ||||||||
| 175 | Functional Genomics Lab Operational Model | NIH investigators are eligible to collaborate with the Functional Genomics Lab staff at NCATS and access screening resources. Genome-wide siRNA screens for humans and mice are available. Also routinely included in screens are microRNA mimic and inhibitor libraries. The Functional Genomics Lab provides in-kind resources in the form of screening support for NIH investigators whose proposals are accepted following internal Institute or Center review as well as a trans-NIH review committee’s approval to proceed. Rejected proposals can be revised to address key concerns and resubmitted for additional review. Following acceptance, the NIH principal investigator and NCATS staff work together to develop a project plan and begin work. For more information, review the latest version of the Assay Guidance Manual. | ||||||||
| 174 | Functional Genomics Lab Goals | The Functional Genomics Lab efforts over the past five years have made a significant impact on the NIH Intramural Research Program (IRP). However, RNAi screening has its limitations, and newer technologies, in particular CRISPR/Cas9-based gene-editing approaches, can help address some of these limitations. The goals of the Functional Genomics Lab are to: Collaborate with NIH investigators to perform genome-wide and targeted RNAi and CRISPR screening projects (assay development, screening and validation) to: Understand fundamental biological mechanisms. Accelerate target discovery for therapeutic development. Develop methods that advance the science of functional genomics screening and informatics. Perform education and outreach to increase awareness of TNRF tools and methods. Pursue new and complementary technologies for exploring gene function. Thus far, CRISPR screens have been performed primarily within an individual laboratory setting, where expertise exists — for example, when employing a pooled library approach with a simple phenotype such as cell survival. However, there is an increasing need for NIH IRP investigators to access robust, reproducible and flexible CRISPR/Cas9-based high-throughput screening (HTS) workflows that are compatible with the assessment of more complex phenotypic assays. Technical challenges associated with the development and implementation of these more complex screens are a barrier for most individual laboratories. Investment in CRISPR/Cas9-based screening platforms within the Functional Genomics Lab will enable the NIH IRP community to access state-of-the-art functional genomic resources beyond RNAi. Learn more about the work of the Functional Genomics Lab in action. | Functional Genomics Lab Goals | Functional Genomics Lab Goals | ||||||
| 173 | About the Functional Genomics Lab | The Functional Genomics Lab, previously known as the Trans-NIH RNAi Facility (TNRF) and administered by staff in NCATS’ Division of Preclinical Innovation, is designed to help NIH investigators use the latest functional genomics technology to advance drug discovery and scientific knowledge about health and disease. About the Technology Small interfering RNA (siRNA) and short hairpin RNA (shRNA) molecules are pieces of RNA that block the activity of genes through a natural process called RNA interference (RNAi). This process has emerged as a powerful tool used in thousands of labs worldwide to understand gene function. Because each RNA molecule can block a different gene, RNAi can tell scientists about the role of any gene in maintaining health or causing disease. In tests called genome-wide and targeted RNAi screens, scientists use robots to introduce siRNAs and shRNAs into human cells to block the activity of genes. Scientists can use these techniques to understand how genes affect drugs’ effectiveness and how they affect disease processes. Another technology available through the Functional Genomics Lab is CRISPR/Cas9, which stands for clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9. This technique enables researchers to make precise edits to a genome, removing, adding, activating or repressing genes based on desired outcome. This could involve correcting a problem caused by a faulty gene by replacing it with a properly functioning version. About the Lab Functional Genomics Lab includes a robotic platform with integrated, automated devices for conducting all aspects of screening assays, including manipulating chemicals and cells, reading the results and imaging the cells. Offline (non-robotic) devices can perform smaller-scale work, from assay optimization through medium-scale screening. Investigators have the option of using several different siRNA libraries as well as targeted CRISPR and small molecule libraries. For data analysis, the facility offers powerful computational tools. Emerging tools for interrogating gene function (e.g., CRISPR) represent new and complementary screening approaches to RNAi. The Functional Genomics Lab team offers diverse scientific and technical expertise and is heavily invested in exploring these emerging technologies, in terms of both their utility and their pitfalls, similarly to what has been achieved with RNAi, to develop and offer a menu of screening platforms. Complementary use of siRNA, CRISPR and shRNA will ensure that all three technologies remain critical to the success of future functional genomics projects. Learn more about the goals of the Functional Genomics Lab. The Functional Genomics Lab staff work closely with collaborating intramural NIH investigators throughout the planning and execution of each research project. The Functional Genomics Lab experts offer advice and assistance on assay development, screening, data analysis, and follow-up, and they typically serve as co-authors for published results. In addition to collaborations, the Functional Genomics Lab staff work to develop methods that advance the science of RNAi screening and informatics and pursue new technologies for exploring gene function. Learn more about how the Functional Genomics Lab works. | About the Functional Genomics Lab | About the Functional Genomics Lab | ||||||
| 171 | Work with ADST | The ADST team’s work to develop innovative assay and screening technology is collaborative by nature; therefore, we always encourage new partnership opportunities, including disease-specific projects with patient advocacy groups and disease foundations. If you have a specific need related to developing assay and screening methods, work with us! Here’s how: Submit a request to work with NCATS. Contact Manju Swaroop with the following details: A summary of idea/vision Research goals Project needs Your level or area of biology expertise Project Selection. We will evaluate your idea and try to match you with the appropriate expert. Project Initiation, Conduct and Closing. Once we determine if our research goals and needs align, a formal collaboration agreement may be executed, if necessary, and the project may begin. | ||||||||
| 170 | ADST Resources | Assay Design Concepts and Proof-of-Concept TestingThis approach employs multimodal outputs adaptable to a variety of biological targets and pathways and can be used to construct assays that phenocopy genetically inherited diseases.Concentration Response-Based Chemogenomic ProfilingThis methodology for high-confidence primary data sets involves micro-volume (3–5 uL) concentration-response determinations of compound series or small libraries for cell-based or biochemical assays.Collaborative Assembly of Preliminary Validation StudiesThis work with high-throughput screening assays supports grant applications or other types of program needs of extramural collaborators.Chemical/Compound LibrariesApproved drugs for repurposingPharmacologically active compounds for chemical genomic-based target assessmentNovel, high-stereogenic carbon content librariesCollections of culturable pre-fractionated natural product extractsMarine natural product extracts and natural product–inspired synthetic collections (e.g., chemical methodology library development) from academic collaboratorsMolecular target–focused and pathway-annotated chemical librariesEquipment/ToolsDetectors and reagent dispensers for a comprehensive variety of assay techniques and formats, including:NCATS-developed high-throughput screening–compatible coincidence reporter systemsLaser scanning cytometry and automated microscopy-enabled high-content assaysTarget-based, purified-component assaysInformatics Analysis ToolsCommercial, open-source and custom bioinformatics and cheminformatics software and analysis algorithmsMore about informatics at NCATSCollaborationsA broad network of internal and external collaborators and expertise for early-stage translational research | ||||||||
| 169 | Current RDCRN Consortia | The RDCRN is designed to advance medical research on rare diseases by providing support for clinical studies and facilitating collaboration, study enrollment and data sharing.Currently, the RDCRN consists of 20 individual clinical research consortia and a Data Management and Coordinating Center (DMCC). Each consortium focuses on at least three related rare diseases or conditions, participates in multisite studies and actively involves patient advocacy groups as research partners. The DMCC enables uniform high-quality data collection and analysis and facilitates information sharing across the network. This robust data source helps scientists better understand the common elements of rare diseases so they may apply that knowledge to improving diagnosis and treatment for these conditions.On Oct. 3, 2019, NIH announced awards to both existing and new RDCRN consortia. Find more information about the consortia. | The RDCRN is designed to advance medical research on rare diseases by providing support for clinical studies and facilitating collaboration, study enrollment and data sharing. | /sites/default/files/rdcrn_1260x630.jpg | Current RDCRN Consortia | The RDCRN is designed to advance medical research on rare diseases by providing support for clinical studies and facilitating collaboration, study enrollment and data sharing. | /sites/default/files/rdcrn_1260x630.jpg | Current RDCRN Consortia | ||
| 168 | RDCRN Funding Information | Find out how to apply for RDCRN funding and see the latest funding opportunities. Current Funding Announcements RFA-TR-22-029: Basket Clinical Trials of Drugs Targeting Shared Molecular Etiologies in Multiple Rare Diseases (U44 Clinical Trial Required) NOT-TR-22-017: Notice of Intent to Publish a Funding Opportunity Announcement for the Rare Diseases Clinical Research Consortia (RDCRC) for the Rare Diseases Clinical Research Network (RDCRN) NOT-TR-22-016: Notice of Intent to Publish a Funding Opportunity Announcement for Data Management and Coordinating Center (DMCC) for Rare Diseases Clinical Research Network (RDCRN) (U2C Clinical Trial Not Allowed) Expired Funding Announcements The following requests for applications are now closed: NOT-TR-20-006: Notice of Special Interest (NOSI): Availability of Administrative Supplements for the Rare Disease Clinical Research Network (RDCRN) PAR-19-220: Clinical Trial Readiness for Rare Neurological and Neuromuscular Diseases (U01 Clinical Trial Not Allowed) RFA-TR-18-021: Data Management and Coordinating Center (DMCC) for Rare Diseases Clinical Research Network (RDCRN) (U2C Clinical Trial Not Allowed) RFA-TR-18-020: Rare Diseases Clinical Research Consortia (RDCRC) for the Rare Diseases Clinical Research Network (RDCRN) (U54 Clinical Trial Optional) Informational Session for RFA-TR-18-20 and RFA-TR-18-21 (PDF - 1.23MB) NOT-TR-18-019: Notice of Availability of Administrative Supplements for Secondary Analysis of Existing Datasets within the Rare Diseases Clinical Research Network (RDCRN) to Explore Outcomes Relevant to Rare Diseases RFA-TR-13-002: Rare Diseases Clinical Research Consortia (RDCRC) for the Rare Diseases Clinical Research Network (U54) NOT-TR-18-002: Notice of Intent to Re-Issue a Funding Opportunity Announcement (FOA) Rare Diseases Clinical Research Consortia (RDCRC) for Rare Diseases Clinical Research Network (U54) RFA-TR-13-003: Data Management and Coordinating Center (DMCC) for Rare Diseases Clinical Research Network (RDCRN) (U01) NOT-TR-18-003: Notice of Intent to Re-Issue a Funding Opportunity Announcement (FOA) Data Management and Coordinating Center (DMCC) for Rare Diseases Clinical Research Network (RDCRN) (U01) How to Apply View individual funding opportunities for specific directions on how to apply to the RDCRN for funding. For general instructions on applying for NIH grants, visit NIH’s About Grants page. |
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