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.

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 Expertise

Common Seed Analysis to Prioritize Hits and On- or Off-Target Effects

A 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.

Testing Multiple siRNAs to Increase Confidence in Genes with Consensus

Because 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.

Separation of False Positives from True Positives Using C911 Controls

In 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.

Functional Genomics Lab Resources

We 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 Libraries

• Ambion Silencer Select Human Genome-Wide siRNA library targeting ~22,000 genes with three individual siRNAs per gene
• Ambion Silencer Mouse Genome-Wide siRNA library targeting ~17,000 genes with three individual siRNAs per gene
• Dharmacon 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 targets
• microRNA mimic and inhibitor libraries
• Ambion Silencer Select Human Druggable Genome siRNA Library V4
• Dharmacon Human ON-TARGETplus siRNA Transcription Factors Library
• Dharmacon Human ON-TARGETplus Epigenetics siRNA Library
• Ambion Silencer Select Human Ubiquitin 96 siRNA Library

Software and Analysis Tools

• RNAi Data Viewer 

This tool allows users to view plates, check stats, screen data and perform a variety of analyses. Access user data (restricted).

Assays

• Simple phenotypes
• Primary reporter assays 
• Complex phenotypes

Robotic Platform

• Liquid handlers, washers and dispensers
• EnVision and Pharastar​ Multilabel Reader (PerkinElmer)
  • Measures luminescence, fluorescence, fluorescence polarization, absorbance and time-resolved fluorescence
• ImageXpress Micro XL confocal high-content imager (Molecular Devices)
  • Automated microscope used for the acquisition and analysis of cell-based images in relatively high throughput