RNAi is a powerful tool for elucidating gene function, especially when conducted at a whole-genome scale and in a quantitative context. Following these tips can ensure an RNAi screen can be conducted quickly, economically, and in a manner that the data can be readily interpreted.
Although lentiviral particles produced using the appropriate packaging plasmids are replication incompetent, it is recommended that they be treated as Risc Group Level 2 (RGL-2) organisms for laboratory handling. Also, use extra caution with lentiviral particles that express shRNAs targeting both cell cycle control and tumor suppressor genes.
Determining the viral titer is critical to ensure consistent transduction conditions within a screen and from one experiment to the next. An indirect titering procedure such as measuring p24 viral protein is reliable only if an optimized and standardized viral production procedure is followed. To fully understand how well the lentivirus will transduce a cell line of interest, one must conduct a functional titering assay such as the colony forming unit (cfu) assay.
Cells transduced with an “empty” (no shRNA construct) or a nontargeting shRNA should be included in all experiments as controls. Such controls help account for any changes in gene expression that might occur due to passaging, treatments, and selection.
When conducting screens, using a redundant shRNA library, which contains multiple shRNA sequences targeting the same mRNA, helps mitigate the potential complication of off-target effects.
When conducting pooled-shRNA screens, it is ideal to set up transductions at an MOI < 1 to ensure that cells are transduced with a single shRNA-encoding viral particle. This allows for the shRNA responsible for a phenotype of interest to be identified during downstream deconvolution.
Andrea Spencer is a senior scientist at Sigma Life Science. To learn more about gene silencing reagents (for siRNA, esiRNA, shRNA, and miRNA), click here.