Variable Dose Analysis: A Novel High-throughput RNAi Screening Method for Drosophila Cells.

ABSTRACT

Genetic screens are a powerful approach to identify previously uncharacterized genes involved in specific biological processes. Several technologies have been developed for high-throughput screens using reagents such as RNAi or CRISPR, and each approach is associated with specific advantages and disadvantages. Variable Dose Analysis (VDA), is an RNAi-based method developed in Drosophila cells that improves signal-to-noise ratio compared to previous methods. VDA assays are performed by co-transfecting cells with a plasmid expressing shRNA, (a type of RNAi that can be easily expressed from a DNA plasmid) against a gene of interest and a second plasmid expressing a fluorescent reporter protein. Fluorescent protein expression, can be used as an indirect readout of shRNA expression and therefore target gene knockdown efficiency. Using this approach, we can measure phenotypes over a range of knockdown efficiencies in a single sample. When applied to genetic interaction screens, VDA results in improved consistency between screens and reliable detection of known interactions. Furthermore, because phenotypes are analyzed over a range of target gene knockdown efficiencies, VDA allows the detection of phenotypes and genetic interactions involving essential genes at sub-lethal knockdown efficiency. This therefore represents a powerful approach to high-throughput screening applicable to a wide range of biological questions.