Table 3.
Methods for experimental verification of RNAi screen results
| Method | Examples | Rationale |
|---|---|---|
| Retest the reagents with the same assay | Test several replicates (including a re-synthesized or new batch of reagent); test single reagents in arrayed format after a pooled approach | Reagent-level verification |
| Retest with a related assay and/or different cell type | Switch the reporters in a dual-reporter assay; test a different cell line, marker, or antibody; test in a different cell line | Reagent-level verification |
| Retest with unique reagents | Test reagents designed to target different regions of the gene | Gene-level verification (confidence increases when more than one works) |
| Assay small molecule(s) | Test a known inhibitor of the gene product in the assay; test small molecules in parallel with RNAi and compare pathways implicated in each | Gene-level verification (correlation is suggestive of an on-target effect) |
| Determine mRNA or protein levels in the presence of the RNAi reagent | Q-PCR or immunoblottinga | Gene-level verification (correlation between knockdown and phenotype is suggestive of an on-target effect) |
| Rescue in the presence of the RNAi reagentb | Test rescue with a genomic fragment, cDNA, or open reading frame construct that evades RNAi knockdown | Gene-level verification (rescue demonstrates an on-target effect) |
| Pattern of gene expression of mRNAs corresponding to hits | Q-PCR or microarray in specific cell types, stages, and/or tissues | Gene-level verification (expression in relevant tissues or stages is suggestive of a relevant finding) |
| Pattern of expression of the proteins corresponding to hits | Immunoblotting in specific cell types, stages, and/or tissues | Gene-level verification (expression in relevant tissues or stages is suggestive of a relevant finding) |
| Subcellular distribution of proteins corresponding to hits | GFP-tagged construct or immunofluorescence | Gene-level verification (expression in relevant subcellular compartments is indicative of a relevant finding) |
| RNAi-induced phenotype in another species | Test effect of knockdown of homologs in mammalian cells as a follow-up to a nonmammalian cell screen | Gene-level verification (similar phenotype provides compelling evidence of a biologically relevant finding) |
| Correlation with a related disease or disorder | Map disease-associated regions, mutations, and amplifications | Gene- and pathway-level verification (disease association is indicative of a relevant finding) |
| Protein-protein interactions | Coimmunoprecipitation, mass spectrometry, yeast two-hybrid screen | Gene- and pathway-level verification (physical interactions among newly identified proteins or between new and established players are indicative of a relevant finding) |
| Genetic analysis in vivo | Test effects of mutations of gene hits in whole animals (same or different species than primary screen cells) | Gene and pathway-level verification [related phenotype provides compelling evidence of a relevant effect and can help refine the role(s) of the genes in specific pathways, events, or behaviors] |
a
Abbreviation: GFP, green fluorescent protein; Q-PCR, Quantitative Reverse Transcriptase PCR.
b
The “gold standard” approach to verification of an RNAi result at the gene level; similar to a genetic test for complementation.