Section14:Introduction

From Assay Guidance Wiki

RNA interference (RNAi) is a mechanism for RNA-guided regulation of gene expression in which short double-stranded RNA (dsRNA) mediates the inhibition of gene expression by hybridizing to a complementary sequence in the targeted mRNA and facilitating transcript degradation or translation inhibition (Figure 1). RNAi can occur endogenously through the expression of microRNA (miRNA) or exogenously via the transfection of synthetic small interfering RNA (siRNA) into cells. Another way to deliver siRNA into the cells is by encoding it in a shRNA expression construct that can be packaged into lentivirus for efficient infection of many cell types.

Experimental RNAi has emerged as a powerful technology to carry out targeted knockdown of gene function. This has been exploited in various drug discovery and development applications, including target characterization and validation, by enabling target gene expression reduction and concomitant measurement of impact on functional readouts. The availability of reagent libraries has paved the way for genome-wide high throughput screens. These screens are generally loss-of-function (LOF) screens that generate ‘hits’ which affect phenotype upon depletion. A common variation of a LOF screen is a synthetic lethality (or, synthetic lethal) screen which combines the use of RNAi and a drug (at single concentration or multiple concentrations) to identify knockdown events that would modulate drug response such as sensitizers that enhance drug effect (see section C). Several recent reviews cover the RNAi biology, experimental parameters and considerations for performing screens (Ref 5-8, 11, 13-15).

In the following sections, we have compiled our learnings around RNAi-based LOF screens of varying scale with an oncology disease focus, to offer a few guidelines on best practices. This is a relatively novel area and this chapter is likely to benefit from growing expertise and improvements in technology and methods.