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RNAi/CRISPR High Throughput Screening Facility

The RNAi/CRISPR High Throughput Screening Facility located at the Greehey Children’s Cancer Research Institute (Greehey CCRI) was founded in November 2016. RNAi screening has developed into a valuable tool for discerning pathways involved in biological processes as a well as the role that individual genes play in specific cellular events. In cell-based assays that model various pathological conditions such as cancer, RNAi screening has become a powerful tool to identify specific gene modulators and targets for drug discovery.

The Greehey CCRI RNAi/CRISPR High Throughput Screening Facility’s goal is to collaborate with UT Health as well as with external investigators to conduct genome-wide RNAi screening projects, including assay development, screening, and gene validation, to understand functional biology of cancer and accelerate target discovery.

In addition, the RNAi/CRISPR Facility is also integrated with other core facilities at UT Health with the aim of adding additional breadth to the research directions of investigators who use the RNAi/CRISPR Screening facility: Computational Biology and BioInformatics Initiative, Genome Sequencing Facility and the Center for Innovative Drug Discovery at UT Health, UTSA.

 

The Greehey CCRI RNAi/CRISPR High Throughput Screening Facility:

The Greehey CCRI RNAi/CRISPR High Throughput Screening Facility’s mission is to expand our understanding of disease mechanisms and identify new targets for therapeutic intervention using genome-wide RNAi screening.

 

 


The Greehey CCRI RNAi/CRISPR High Throughput Screening Facility is located in 1000 square feet of lab space and has a separate tissue culture room in the Greehey CCRI building. The RNAi/CRISPR facility has all the necessary equipment and personnel to run automated arrayed whole genome siRNA and miRNA screens using high content imaging or plate-based fluorescence or luminescence formats.

The facility also has the reagents and data analysis tools for performing screens using human pooled lentiviral CRISPR libraries. The facility is equipped with a Vala Sciences IC200-KIC high content imaging system which is robotically integrated with a Liconic microplate incubator for live cell as well as fixed cell imaging. The BMG Labtech Pherastar FS multimode plate reader can accommodate any fluorescence or luminescence assay format. For rapid processing of screening pates, we use a LabCyte Echo acoustic dispensing system for plating library oligos into 384-well microplates. Reagents, buffers and cells are dispensed using a Tecan Freedom EVO 150 and a Bio-Tek EL406 bulk dispenser. The RNAi/CRISPR Facility has optimized RNAi HTS in 15 different pediatric cancer cell lines and as investigators needs are addressed, we are continuing to update that list of cell lines.


  • Consultation and hands-on support on assay design (crucial step in HTS that will dictate the biological relevance of the genes identified).
  • Biological assay optimization.
  • Development, testing and screening of RNAi libraries using cell-based high-content imaging, and cell-based multimode plate reader readouts.
  • Support statistics and bioinformatic analyses.
  • Support validation of candidate gene targets.
  • Assessment and identification of druggable targets.

After submission of a Project Application form, costs of the various screening formats will be discussed with Dr. Hart.


 

Automated Labcyte Echo Acoustic Dispenser

  • Nanoliter-scale reagent dispenser
  • Tipless transfer, resulting in dramatic cost savings
  • Improved information about sample integrity and improved transfer quality through measurement of DMSO or aqueous buffer levels.
  • Elimination of RNAi or compound loss, cross-contamination, carryover or leachates through non-contact transfer.
  • Extend RNAi library life through miniaturization of assays.
  • Greatly simplified assay development and dilution processes through the flexibility to transfer any volume of liquid from any well to any other well.
  • Robotic integration with 80 microplate ambient storage units.
  • Robotic integration with Agilent PlateLoc heat sealer, Brooks x-Peel desealer and Agilent Barcode microplate labeler.

 

Vala Sciences IC200-KIC Automated High Content Imaging System.

  • 5.54Mp sCMOS camera (100fps, full frame)
  • Plan APO 4X-60X objectives
  • Rapid autofocus (<200ms)on every field
  • Simple optical path
  • High-resolution positioning (<100nm)
  • Solid state light engine with 5 discrete bands
  • Filter set (DAPI, FITC, TRITC, Cy5)
  • Environmental control (temperature, CO2, N2)
  • Structured Illumination Microscopy module
  • Integrated with a PreciseFlex 400 sample handler and a Liconic STX44 incubator for live cell experiments.
  • Image analyses includes cell counting, live/dead assays, subcellular localization, signal transduction pathway analysis, cell cycle, cell morphological analyses, cell migration

 

BMG Labtech Pherastar FS multimode plate reader:

Tecan Freedom Evo Pipetting Station

  • Compound plating and reagent dispensing.
  • Volumes ranging from 1.0 µl to 70 µl/well into multiple plates (96- and 384-well microplates
  • Contained within a NuAire HEPA filtered enclosure

 

Bulk dispensing and plate washing for biochemical and cell-based assays

BioTek EL406 dual bulk dispenser/microplate washer:

  • Low-volume dispensing and plate washing.
  • Especially designed for gentle washing of microplates containing living cells.
  • Integrated with a BioStack microplate stacker.

General Lab Facilities for assay preparation:

  • Cell culture incubators under normoxic and hypoxic conditions
  • Cell culture hoods
  • Microfuges and centrifuges
  • Liquid nitrogen cryopreservation
  • Refrigerators and freezers

RNAi Libraries

Human Ambion Silencer® Select siRNA Libraries

Genome-wide siRNALibraries

Genes siRNAs
Human Genome siRNA Library V4 21584 64752

 

mirVana miRNA mimic and inhibitor Libraries

Targets mirBase
mirVana miRNA Mimi Library 2555 v20
mirVana miRNA inhibitor Library 2555 v20

Defined siRNA human subsets available for screening:

  • Epigenetics
  • Drug Targets
  • DNA Damage
  • Oncogenes
  • Tumor Suppressors
  • Cell Surface Receptors
  • Kinases
  • Phosphatases
  • Membrane trafficking
  • Ubiquitinylation
  • Ion Channels
  • Nuclear Hormone Receptors
  • Proteases
  • transcription Factors

 

Pooled CRISPR Screening

The facility pooled CRISPR libraries include the Human GeCKO v2 which contains 123,411 lentiviral sgRNA vectors targeting 19,050 genes (Shalem et al , Nat. Methods 2014) and the Human Synergistic Activation Mediator (SAM) which contains 70,290 lentiviral sgRNA vectors targeting 23,340 transcript isoforms for gain-of-function screening (Konermann et al, Nature 2015). Prior to applying negative or positive selection pressure, the baseline sgRNA distribution needs to be determined. This baseline determination will be used to identify enrichment or deletion of specific sgRNAS. As in a synthetic lethality screen, the identification of candidate genes is based on frequency of sgRNA distribution which is determined through the use of next generation sequencing approaches.

Following selection pressures of 7 and 14 days and the determination of the frequency of sgRNAs, distribution of sgRNAs is determined using the RNAi Gene Enrichment Ranking (RIGER) method. As with RNAi assay results, the ranked genes are then analyzed using software that has been developed by the UTHSCSA Computational Biology and Bioinformatics Initiative to perform systems biology and pathway mapping analyses.


Data management, analysis and Bioinformatics:

The Tibco Spotfire software suite which is an integrated solution for high throughput screening data analysis serves as our platform for data analysis and hit selection. Spotfire allows us to analyze and supervise the HTS data using multivariant and multiparametric outcomes to eliminate systematic errors and to carefully select promising candidates for further study. Using positive and negative controls, data quality and variability for each of the measures are monitored per plate. Data are also normalized across plates. Hit selection is performed on a per plate and per screen basis. We use the following statistical measures for hit selection: z* score, strictly standardized mean difference (SSMD) and t-statistic. The data from these analyses are integrated with software at the Computational Biology and Bioinformatics Initiative, located at the GCCRI and directed by Dr. Yidong Chen, to perform systems biology and pathway mapping analyses.

 


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