Genome-wide RNAi Screening and Analysis (GRSA) Shared Resource
(Thomas Westbrook, Ph.D., Director;
Dan Liu, Ph.D., Co-Director)
Genetic screens have been a powerful approach for defining signaling and developmental pathways in model organisms. RNA interference (RNAi) represents a robust technology for loss-of-function genetics in human and mouse systems. The Genome-wide RNAi Screening and Analysis (GRSA) Shared Resource was established to facilitate investigators in their use of new RNAi technologies and genetic screening methods, as well as cutting-edge technology to probe important mechanistic questions in basic and translational cancer biology.
RNAi vector resources
GRSA maintains the GIPZ collections of short-hairpin RNA (shRNA) vectors (from Open Biosystems) targeting nearly all of the protein-coding genes in the human and mouse genomes.
1. Individual vectors. DLDCC investigators can obtain any individual shRNA vector from human and mouse GIPZ shRNA libraries via the Shared Resource. Format of individual lentiviral shRNA vectors provided includes bacterial cultures, purified plasmid DNA, or lentiviral supernatant.
2. Pre-arrayed sublibraries. GRSA also utilizes robotic instrumentation to assemble Gene-Ontology (GO) or custom shRNA vector collections based on investigator requests. These collections are assembled in multi-well plates or as pools, and distributed in bacterial, DNA, or lentiviral formats. Current GO sublibraries include shRNA collections against kinases, phosphatases, E3 ligases, and G-protein coupled receptors.
3. Custom libraries. Investigators may request assembly of custom shRNA libraries (in bacterial glycerol stocks, 96-well plates) tailored to their specific needs.
Genome-wide genetic screens
GRSA assists and performs cell-based screens utilizing either the shRNA libraries and/or reagents provided by individual investigators (e.g., chemical or siRNA libraries). Screens may be carried out in 96-well format (well-by-well) or in pools. GRSA also offers both high-throughput flow cytometry or automated inverted fluorescence microscopy for functional and phenotypic analysis in 96-well based screens. Typical workflow includes:
(a) Assay development (consultation, screening methods optimization, etc)
(b) Target library assembly (in 96-well plates or pools; includes cherry picking, DNA purification, etc.)
(c) Lentivirus production and infection
(d) Phenotypic analysis (e.g., using image- or flow cytometry based data acquisition)
(e) Large-scale data processing, analysis, and storage
Automated preparation and application of custom shRNA libraries
GRSA offers services for the replication, preparation and subsequent utilization of the shRNA vectors, either as single vectors or as arrayed on 96-well plates.
1. Library manipulation/liquid handling. Examples include shRNA vector “cherry-picking”, library replication for archiving, and shRNA library re-arraying (e.g., from 96 to 384-well plates) to increase throughput.
2. Automated nucleic-acid purification. DNA plasmids encoding the shRNA sequences are robotically prepared in 96-well format.
3. Automated mammalian cell transfection. DNA plasmids prepared in 96-well plates are transfected into mammalian cells robotically in 96-well format.
4. Individual or high-throughput lentiviral production and infection. GRSA prepares lentivirus stocks from either single shRNA vectors or vectors arrayed in 96-well plates.
High-throughput phenotypic analysis
1. Automated fluorescence microscopy. GRSA houses an automated inverted fluorescence microscope (Micro) and an automated confocal fluorescence microscope (Ultra) (Molecular Devices). Both microscopes utilize high-speed laser autofocus and custom 4x-100x optics, and can scan 6- to 1536-well microtiter plates. Micro is equipped with environmental control (CO2, temperature, and humidity), while Ultra features four lasers that allow simultaneous scanning.
2. High-throughput flow cytometer. GRSA offers high-throughput flow cytometry analysis through the LSRII bench-top flow cytometer (BD Biosciences). It is configured with the BD Biosciences blue laser system and equipped with the High-throughput Sampler (HTS) for 96-well plate based data acquisition. The LSRII-HTS has the capacity to process 4 plates (96-well) per hour.
GRSA offers four workstations and several software packages for image- and cytometry-based data analysis. The imaging software MetaXpress provides tools for processing and analyzing high-content cellular images, including over 18 application-specific software modules for the specialized image segmentation and quantitation needs of screening assays. Examples include cell cycle analysis, granularity, multi-wavelength cell scoring, and multi-wavelength transflur. AcuityXpress is a cellular informatic software specifically designed to address the needs of high-content imaging data analysis, and seamlessly integrates images with analysis data output. The FacsDIVA software enables automated data acquisition and analysis of large sample size. FlowJo facilitates complex data analysis and is especially suitable for automated repetitive data processing.
Project management and data storage
To integrate the use of RNAi libraries, robotics, flow cytometry, and high throughput microscopy to enable genome-wide genetic screens, the GRSA Shared Resource has constructed an information technology framework to support and facilitate automated analysis of the data, including the Storage Area Network (SAN).
Charge Back Fees and Forms
• Pricing for services can be found here.
• For GIPZ shRNA vector request, please go to www.bcm.edu/cbass for detailed instructions. For all other services, please fill out the form and email to firstname.lastname@example.org
• For instrument use, please go to http://www.cbass.bcm.tmc.edu/Reservations/
• For all inquiries, please email us at email@example.com
Thomas Westbrook, Ph.D.
Dan Liu, Ph.D.,
Richard Sucgang, Ph.D.
Jun Xu, Ph.D.
One Baylor Plaza,304B
Cullen Research Building
Baylor College of Medicine
Houston, TX 77030
For more detailed information regarding the services provided, account setup, and other resources available at GRSA, please visit our BCM-wide page (www.bcm.edu/cbass).