RNA interference (RNAi) is a method that is based on an endogenous cellular response to the presence of long or short double-stranded RNA and is useful to knock down (i.e. reduce but not eliminate) gene function. The specific RNAi reagent and the method for its delivery to cells is likely to be different for different types of cells, model organisms and assays. But in general, a gene-specific segment of double-stranded RNA is introduced into cells, leading over time to degradation of the endogenous target mRNA. Short segments (~21 bps) are typical for mammalian systems, as longer segments can induce a non-specific interferon response. Longer segments (~500 bps) are typical for model systems that lack an interferon response, such as Drosophila. Similarly, the appropriate delivery systems differ for different systems. Common systems for delivery of the RNAi reagents include viral transduction (shRNAs), transfection or electroporation (shRNAs, siRNAs or dsRNAs) and bathing (dsRNAs). RNAi can be compared with loss-of-function genetic approaches. When the normal function of a gene is required for a given function, RNAi knockdown may lead to a phenotype detectable in an assay that tests that function, either directly or indirectly. Example assays include activation of a transcriptional reporter; responses to an external stimulus (e.g. stress, drug treatment, pathogen); and sub-cellular localization or morphology of a specific protein or organelle. Cell-based RNAi screens are typically performed in one of two formats, pooled or arrayed. Both formats are supported by the collaborative DF/HCC Collaborative Functional Genomics Core.
(1) With a pooled approach, all of the gene-specific reagents are pooled together (or synthesized en masse) and added, at random, to cells. This could be compared to transformation of a cDNA library into bacterial cells. You will know that each cell gets about one gene-specific RNAi reagent. But you will not know which cell got which one. Pooled Selection. In some cases, the researcher next performs a selection; that is, applies a treatment (e.g. a drug, condition or pathogen) that kills most cells, leaving only cells that have an appropriate RNAi knockdown to survive. That is, when the gene-specific RNAi phenotype is viability under conditions in which most cells would die. Subsequent to the selection, a molecular method is used to figure out which RNAi reagent (and thus, which putative gene target) is responsible for “escape” of the lethal treatment.Pooled Comparison. In other cases, sub-sets of cells (or different cell types) are treated differently before and/or after being subjected to the RNAi library (creating reference and experimental sets). Subsequently, a molecular method such as micro-array analysis is used to compare the two sample sets. The array detects the abundance of the RNAi reagents (such as via detection of the shRNA sequence itself or a molecular barcode in the construct). The array is used to determine which RNAi reagents (thus, which putative gene targets) are under- and/or over-represented in the experimental set as compared with the reference set.
Advantages: a pooled approach is typically more practical to do in a standard lab space and in a relatively small total volume, thus making it more feasible for some groups and less expensive.
Disadvantages: deconvolution of the putative “hits” (positive results) requires specialized or custom microarrays and their analysis.
(2) With an arrayed approach, each unique RNAi reagent (or unique set of reagents targeting a single gene, such as for a small pool of independent siRNAs targeting one gene) occupies a unique well in a microtiter plate, such as a 384-well plate. Experiments are done in 384- or 96-well format. Thus, after the assay, you can easily determine which cells got which specific RNAi reagent by looking up the identity of the reagent in a given well using a database or spreadsheet.
An arrayed approach is useful for a wide variety of cell-based assays, including plate-reader assays (i.e. total light output, such as from GFP, FITC or luciferase) and low- or high-content image-based assays. Most cells will have the expected normal response, behavior, shape etc. But some specific reagents will have an abnormal response, behavior, shape etc. (mutant phenotype) that is detected in the assay. For some approaches, such as an imaging approach, multiple read-outs might be detected (i.e. via antibodies or dyes, using multiple fluorescent channels).
Advantages: Arrayed screening arguably opens the door to the widest possible range of cell-based assays that can be performed. You will be able to quickly associate hits with the putative gene target of the unique RNAi reagent that was present in a specific well in which the mutant phenotype was observed.
Disadvantages: Typically requires specialized high-throughput equipment for plate processing and assay detection and reagents, media, etc. can be costly.
We recommend also reading the screening guidelines at the ICCB Longwood Screening Facility web site.