Screening of Chemical Compounds or siRNA Libraries
The UT Southwestern High Throughput Screening Core works collaboratively with principal investigators on both chemical and genome-wide siRNA Screens. We also offer access to an arrayed CRISPR library for follow up to small molecule and RNAi screens as well as for tailoring a focused CRISPR screen to a particular biological or disease-related question. In each case, a project begins with a discussion between the principal investigator and the Core director, Dr. Bruce Posner. We cover the goals of the screening project as well as the associated assays and proof-of-concept studies. If the project is within the scope of the Core’s mission and capabilities, then a scientist from the Core is assigned to work as a point of contact (POC) for the scientist spearheading the project in the principal investigator’s lab. Together, they optimize the assays for robustness and reproducibility (For more details, please see “Assay Development” under this header.). Once the assays for the project are ready to go, then screening ensues. See other sections under this heading (“Chemical Pilot Screens” & “Large Screens”) for more details of Core support and capabilities.
For details regarding access to run small or large screening projects as well as advanced projects with the Core (chemical tool development or pre-clinical drug discovery project support), please see the corresponding sections under “Access and Support.
- Assay Development
Assay development is conceptually similar for RNAi and compound screens. In both cases, the primary assay must reflect an appropriate biological context and be sufficiently robust to detect desired changes in the biological response when exposed to a genetic or chemical perturbagen.
Dr. Posner and the HTS staff will comment on the practicality of the assay, offer suggestions for counter and secondary assays, and, if needed, provide alternative assay approaches that have proved effective in previous projects. The HTS Core has more than 40 years' experience in assay development and screen execution and is a valued collaborator in these aspects of HTS.
Both HTS Core and project scientists work collaboratively to develop, refine, and validate parameters and controls (positive, negative, and neutral) for the primary assay such that it is robust (Z values > 0.5 over many assays and experimental days), tolerant of effects from DMSO, free from systematic effects (e.g., plating artifacts, liquid handling errors, etc.), simple (most assays have less than three liquid additions and are endpoint assays), and efficient in use of reagents, HTS equipment, personnel, and resources. Secondary assays are developed in parallel to the primary assay and must meet the same criteria. RNAi screens are typically run in 96-well microtiter plates (three replicates per siRNA pool), while compounds screens are run in a 384-well format (one replicate per compound).
Once an assay has an acceptable Z’ in small-scale experiments for chemical or RNAi screen, it is tested in three to 10 plates treated under "mock" conditions, an experiment in which the HTS protocol is executed but no actual library samples are tested. In the case of compound screening, DMSO is substituted for the chemical library in the test wells (columns 3 to 22 of a 384-well plate) and for RNAi screening, transfection reagent plus a non-targeting siRNA or transfection reagent alone is substituted for the library in columns 2 to 11 of a 96-well plate.
Assays that are reproducible and largely free of plate or systematic effects are deemed optimized and ready for screening, provided approval has been obtained from the Core Director (small screening projects) or the HTS Oversight Committee (large screening projects).
- Chemical Pilot Screens
For compound screens, a pilot experiment can be done with an 8,000-compound diversity subset. This subset is an approximate representation of the chemical space in the larger 300,000+ UT Southwestern small-molecule commercial library.
Principal investigators are required to dedicate a scientist to work on the HTS screen. This person will work with the HTS scientific staff, who will operate the robotic liquid handlers and perform all operations involving the dispensing of compounds from library plates to experimental plates. The HTS staff scientists will also read the plates for each experimental run, analyze the data, and provide a report to the principal investigator and his/her scientist leading the screening effort.
Following the primary screen, the principal investigator can opt to cherry-pick hits for confirmation studies using the primary assay (three replicates per compound) and further characterization using secondary and counter screens. The PI is supplied with 4 μL of a 5 mM stock of each cherry picked compound. This is enough for several plate-based assays in a limited dose-response format, if desired.
- Large Screens
Results from assay optimization, mock screens, and pilot screens (if applicable) can be used as preliminary data in the proposal for a large genome-wide siRNA screen or a full-library chemical screen. Once the Oversight Committee approves a large screening project, the HTS Core will schedule several experimental runs (25-50 plates per day) for the screen over an eight- to 12-week period. The scientist from the principal investigator's lab will participate in screen execution while HTS scientists will dispense the library, collect assay data, monitor screen quality, analyze screening data, and provide regular reports of screen progress.
At the conclusion of the screen, the HTS Core will provide a summary of the primary data for the screen. For chemical screens, principal investigators can select 1,920 compounds for cherry-picking and follow-up studies. The Core provides 4 μL of a 5 mM stock solution for each cherry-picked compound. This is more than sufficient to carry out confirmation studies with the primary assay and additional follow-up studies that can include secondary assays and counter screens.
For follow up studies in RNAi screens, the HTS Core will provide the ordering information for commercial siRNA libraries and the investigator can order primary hits that are of interest. The HTS team can provide follow-up support (e.g., inventory management, assay execution, data analysis, etc.) for siRNA screens, if needed.