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 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 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 particular gene modulators and targets for drug discovery.
The goal of the Greehey CCRI RNAi/CRISPR High Throughput Screening Facility 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 the 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 San Antonio and UTSA.
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 a 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 the 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.
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 select promising candidates for further study carefully. 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 Greehey CCRI and directed by Dr. Yidong Chen, to perform systems biology and pathway mapping analyses.