For example, it is not helpful to select a very strong positive control that yields a high-quality Z'-factor if it is not comparable to the strength of the expected hits sought in an actual screen. Good judgment should instead prevail, and positive controls should be selected based on the intensity of the hits hoped to find. This is often the result of undue emphasis on minimum criteria for acceptance for screening, such as a Z'-factor cutoff. In selecting controls, there is a temptation to select reagents with very strong effects as positive controls. Although differences in modality may complicate direct quantitative analysis of such controls, such 'artificial' controls are often helpful during assay development and optimization, and provide a sense of the dynamic range to be expected in the screen. For example, expression of a constitutively active form of a tagged protein or knockdown of a target by RNAi can simulate the effects of a sought-after small molecule in a screen. However, any reagent that induces the phenotypic change of interest can serve as a positive control if necessary, even if artificial. a small molecule control for a small molecule screen, and an RNAi-based control for an RNAi screen). Ideally, a positive control is of the same type as the reagents to be screened (e.g. Long running assays will typically accumulate positive controls over time and may even change the perceived limits or dynamic range of the assay. However, these may not be known in advance. Once a condition is identified and demonstrates such a measurable change, then it can serve as a positive control going forward.įor profiling assays, in which a large variety of cellular features are measured to identify similarities among samples, and hence designed to have multiple readouts, several different positive controls for each desired class of outcomes may be necessary. In these situations, an assay measuring a real biological process may still show a phenotype of interest under some conditions that can be observed and measured even if positive controls that induce high levels of cells with the phenotype do not exist. However, positive controls may not be readily available. Using controls is required to calculate a performance envelope for measuring the assay quality and phenotype feature space (see ' Assay Quality and Acceptance Criteria for HCS' section below).

Whenever possible, positive and negative controls should be included in an assay.

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