Phenotypic assay design

Other assays typically employed in the course of a drug design project include physicochemical binding assays (based on, for example, NMR or Biacore surface plasmon resonance methodologies), mode-of-action cell assays (e.g., employing a specific antibody-based biomarker readout of the target or a downstream target), and phenotypic assays (e.g., cell proliferation assays). 

After the selection of the specific techniques to be employed, one must determine how to quantify the genes associated with the phenotype of interest. The design of an assay that acts as a reporter or a molecular sensor can prove to be the most challenging step in designing a successful RNAi screen. The assay must be both highly reproducible and sensitive, without noise, and exhibiting minimal standard error. Traditional assay development techniques must be taken into account for all RNAi screens as they are for conventional compound-based screens. 

The excessive number of animals per test group is also pointed out and tackled with recommendations on the reduction of animal numbers. In particular, a better use of statistics in the design of experiments is recommended in order to reach an optimal compromise between animal number and variability of the results. The use of homogenous populations is advocated as a means to minimize interindividual variability if physiological variation between individual animals can be controlled, and statistical methods used to exploit this control to the full, the number of animals necessary for assay purposes can be dramatically reduced. This results, for instance, in the use of only one breed of rats for one set of tests, rather than a mixture of different breeds with the objective of mimicking the phenotypic variability of humans. This latter approach would result mainly on the study of the differences between breeds rather than the actual effects of the compound. 

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