Assay Development

Assay development is conceptually similar to 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 of 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 the 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 compound 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).