Setting Up a Screen
Steps to screen development:
- Assay Development- Design the experimental parameters
- Pilot Screen- Initial run through and evaluation of the assay using a portion of the library
- Primary Screen- Screen all of the compounds/siRNAs in the library
- Secondary Screen- Further Screen of hits from primary screen.
- 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.