Setting Up a Screen

Steps to screen development:

  1. Assay Development-  Design the experimental parameters
  2. Pilot Screen- Initial run through and evaluation of the assay using a portion of the library
  3. Primary Screen- Screen all of the compounds/siRNAs in the library
  4. Secondary Screen- Further Screen of hits from primary screen.
  5. Tertiary screen/Further Characterization- Characterization of hits confirmed in secondary assay

In order to perform a screen the investigator must have an assay developed for screening. The core can assist in scaling the assay to 96 or 384 well plates.

Properties of a good screen

  • Clearly defined and easily scorable phenotype
  • Low variability
  • Good positive and negative controls
  • Large dynamic range
  • Highly reproducible
  • Low rates of false positives and false negatives
  • Approximately 1-5% of the collection scored as hits

Parameters to consider when designing an Assay:

Compound Library Screens

Cell-based assays

  • Cell line Choice
  • Cell number
  • DMSO side-effects
  • Control Drug(s)
  • Time point(s)
  • Sample processing

In vitro protein assays

  • Protein production, stability, concentration
  • DMSO side-effects
  • Control Drug(s)
  • Concentration of other key reagents/substrates

siRNA Library Screens

  • Cell number
  • Time point(s)
  • Transfection/lipofection protocol
  • Examine statistical parameters of assay


A pilot study is run in the core after assay parameters are optimized. It generally consists of a portion of the library to be screened. The results of the pilot study are examined for the properties of a good screen including hit rate, plate variability, controls, etc. Based on the results the investigator will either reevaluate parameters or decide to continue on to the full-scale primary screen.

A primary screen involves screening the entire library for hits (compounds/siRNAs producing the desired phenotype). Controls are run on each plate to assure that the assay is working correctly. Hits are determined on a plate-by-plate basis using the criteria selected by the investigator. The core has available basic information on the hits such as gene name/function, chemical structure, LogP values, etc.

Secondary screens serve to validate hits obtained in the primary screen and can take several forms. They include things such as using a different cell line or concentration of compound/siRNA or using a kinase dead form of a protein. The core can provide small amounts of chemicals/siRNAs for secondary screens.

Tertiary screens involve further characterization of hits that pass the secondary screen. These are generally done in the investigators lab with compounds/siRNAs purchased by the investigator. Compounds/siRNAs are available for purchase from the original company in differing amounts.