Multiplexed RNAi screens are 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.

Decode™ RNAi viral screening libraries can be used in positive selection screens to identify shRNA in cells that survive some selective pressure because they have a particular gene knocked down or cause a selectable pheotype.  Negative selection (dropout) screens can also be performed to track the relative abundance of shRNA in a complex population of cells. The steps involved in performing a positive and negative selection screen are listed below:

OVERVIEW OF POSITIVE SELECTION SCREENS

Transduce target cells:
Each Decode RNAi Viral Screening Pool kit contains pools of ~10,000 unique shRNAmir in lentiviral particle form.  Cells should be transduced with each pool 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 (Brummelkamp, Fabius et al. 2006; Ngo, Davis et al. 2006), exposure to virus, induction of apoptosis or growth in the absence of extracellular matrix (Westbrook, Martin 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, Martin et al. 2005), selection by FACS, immunobeads (Gazin, Wajapeyee et al. 2007) or another approach whereby the population of cells with the desired phenotype are isolated and collected (Brummelkamp, Fabius et al. 2006).

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 with the Decode RNAi viral pools.

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 provided along with the Decode RNAi viral pools.

OVERVIEW OF NEGATIVE SELECTION SCREENS

Transduce target cells:
As with positive selection screens, cells should be transduced with lentiviral shRNAmir constructs at single copy for negative selection screens. Single copy integrations are necessary in a multiplexed RNAi screen in order to effectively de-convolute the resulting phenotype. Successfully transduced cells are then split into control and experimental plates.

Selective pressure:
The transduced cells are subjected to selective pressure to induce the phenotype of interest.  Negative selection screens are especially useful in identifying shRNA that cause transduced cells to become depleted relative to the population as a whole.  Negative selection screens have been used to identify shRNA that promote drug sensitivity (Schlabach et al. 2008) or cell lethality (Silva et al. 2008).

PCR amplification and labeling:
Genomic DNA is isolated from transduced cells following a selective pressure.  Barcodes corresponding to specific shRNA are amplified by PCR and labeled with either Cy5 (treated) or Cy3 (control) dyes.  Samples are then competitively hybridized to custom microarrays spotted with the unique barcode sequences.

Barcode array analysis:
Microarrays are scanned and analyzed to determine abundance levels of individual shRNA constructs via barcode identification.  By comparing the processed signals at each spot (Cy5/Cy3), shRNA drop outs can be determined.

Additional information from Agilent on using the barcode microarrays
CGH Microarray protocol
Gene Expression protocol
SurePrint Technology
Feature Extraction Software