Multiplexed RNAi screens are very dependent on RNAi triggers that can produce effective knockdown with single copy integrations. Transduction should be performed at multiplicity of infection (MOI) of less than 1 (usually between 0.1-0.3) to ensure that each cell only integrates a single shRNA.
GIPZ lentiviral shRNAmir have been shown to produce effective knockdown at low copy. See
additional details here.
Positive selection screens are used to identify shRNA in cells that survive some selective pressure because they have a particular gene knocked down. Decode™ RNAi Viral Screening Pools are optimized for positive selection screens. The steps involved in performing a positive selection screen are listed below:
Transduce target cells:
Each Decode RNAi Viral Screening Pool kit contains 70,000 unique shRNAmir in seven pools of 10,000 shRNAmir each. Each pool should be transduced at single copy to generate a complex population where each cell expresses a single shRNA producing effective knockdown of the target gene. Single copy integrations are necessary in a multiplexed RNAi screen in order to effectively de-convolute the resulting phenotypes.
Selective pressure:
The transduced cell population is subjected to some selective pressure to induce the phenotype of interest. Selective pressure may include irradiation, drug or small molecule treatment (Ngo et al 2006), exposure to virus, induction of apoptosis or growth in the absence of extracellular matrix (Westbrook et al 2005).
Selection of phenotype:
After exposure to selective pressure, cells with the appropriate phenotype are selected. This may be a growth selection (Westbrook et al 2005), selection by FACS, immunobeads (Gazin et al 2007) or another approach whereby the population of cells with the desired phenotype are isolated and collected.
PCR amplification:
After selecting for the phenotype of interest, the shRNAmir integrated into the surviving cell populations are recovered by PCR. Genomic DNA is isolated from these cells and primers designed to flank the shRNAmir hairpins are used to amplify the shRNAmir sequences. Forward and reverse primers for PCR amplification are provided in the Decode RNAi viral pool kit.
Identification of positive hits:
Hits from the positive selection screen can be identified by sequencing the PCR amplicon recovered from the cells expressing the phenotype of interest. Sequencing primers are also provided in the Decode RNAi viral pool kit.
Published multiplexed RNAi screens performed using Open Biosystems' shRNA libraries
Gazin C et al 2007 "An elaborate pathway required for Ras-mediated epigenetic silencing" Nature Vol 449: 1073-1077.
Schlabach M et al 2008 "Cancer Proliferation Gene Discovery Through Functional Genomics" Science Vol 319, 620-624
