TNRF Resources

TRNF experts at NCATS 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

Examples: viability, cytotoxicity

A microscope image of six live round cells is presented as an example of an assay that explores morphology or cell count.

Assays that explore one aspect of morphology or function/loss of function.

Pathway reporter assays

Examples: luciferase, beta-lactamase

A large, gene network graph is presented as an example of a signaling pathway that can be assayed using a reporter construct.

Assays using a specific read-out mechanism to “report” data, or “reporter assays.”

Complex phenotypes

Examples: high-content imaging, cell cycle, translocation

A microscope image of cells with two fluorescent markers, green and red, designed to study the interaction of two proteins within a cell.

Assays that explore more than one aspect of morphology or function/loss of function, usually a process with two or more steps.

3-D physiologically relevant models

Representation of 2-D and 3-D RNAi invasion screening data. The two panels are shown 24 hours after Matrigel addition. The left panel shows a well with a breast cancer organoid with an siRNA targeting MMP9 (siMMP9). The right panel shows a 3-D organoid targeted with a non-targeting siRNA AN2. These two siRNAs will be used as negative and positive controls for data analysis.

Representation of 2-D and 3-D RNAi invasion screening data. The two panels are shown 24 hours after Matrigel addition. The left panel shows a well with a breast cancer organoid with an siRNA targeting MMP9 (siMMP9). The right panel shows a 3-D organoid targeted with a non-targeting siRNA AN2. These two siRNAs will be used as negative and positive controls for data analysis.

 

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
A schematic cartoon of a high-throughput robotic system that houses instruments required for high-content screening. These instruments include plate-based readers, plate washers and liquid dispensers.

Schematic illustration of the RNAi robotic platform.

Photograph of the RNAi robotic platform.

Photograph of the RNAi robotic platform.