A P P L I C AT I O N N O T E

High content
analysis usingtargeted
RNAi-mediatedgene knockdown.

Introduction
Small interfering RNAs (siRNAs) are able to effectively
down‑regulate gene expression in mammalian cells throughRNA interference (RNAi). Collections of siRNAs targeting genefamilies, biological processes, or even entire genomes providea systems-based approach for identifying the role of particulargenes in a cell. RNA-based gene function analysis is particularly
powerful when combined with high content screening (HCS)
where multi-parametric assays are used to better define
the function of a specific gene target. In this study, parallelknockdown of 80 genes that are known or predicted to beinvolved in the cell cycle regulation was performed using
Dharmacon™ SMARTpool™ reagents. Monitoring of four relevant
phenotypes (cell cycle phase, nuclear area, cell count, andlocalization of phospho-ERK) using a high-content imager toidentify gene targets involved in cell cycle regulation and cell
cycle phase-specific arrest. These results clearly demonstratethat cell cycle status and phenotypic changes in cell populations
can be easily assayed using a highly specific and functional
SMARTpool siRNA library, followed by data acquisition
using high content screening instrumentation and software.
In addition, these results also show the utility of combining
rationally designed SMARTpool reagents and high‑content
imaging technology for functional mapping of genes in specificdisease states or model assays.

High content analysis using targeted RNAi-mediated gene knockdown.

Materials and methods
siRNA library and transfection

High content screening and analysis
The transfected cells were fixed and analyzed for thefollowing cell features: cell cycle phase characterization,The siRNAs used in this study were designed to target

nuclear area measurement, cell count per field, and ERK

80 individual cell cycle-related genes and are a subset

activation. The ERK Activation Thermo Scientific Cellomics™

of the human siGENOME™ siRNA library for cell cycle

HitKit™ HCS Reagent Kit (Cat #K0100071) was used forregulation. The complete library consists of SMARTpool

immunofluorescence staining of phosphorylated ERK (pERK)

siRNA reagents designed to target 111 genes involved inand a DNA specific stain, whose fluorescence is proportional

cell cycle regulation. Each SMARTpool reagent consists

to DNA content. The plates were imaged and quantitativelyof four rationally designed siRNAs, targeting a distinct

analyzed on the ArrayScan VTI HCS Reader using theregion of the target mRNA, thus assuring gene silencing

Cell Cycle and Cytoplasm to Nucleus Bio-Application

and reducing the incidence of off-target effects. A549 cells

image analysis software. Numeric data generated by thewere plated in 96-well microwell plates at a density

ArrayScan VTI HCS Reader were evaluated with the vHCS™

of 5,000 cells per well 24 hours prior to transfection.

Discovery Toolbox and exported to Spotfire™ Decision Site™

Cells were transfected with the SMARTpool siRNA reagents(Spotfire, Inc. Somerville, MA) for further analysis.

(100 nM final concentration) using the DharmaFECT™ 1
at a final concentration of 0.15 μL/well. Transfection wasmonitored by microscopy and acquiring images on theArrayScan VTI HCS Reader (version 5.5), using the siGLO

Results

RISC‑Free™ siRNA (Cat# D-001600-01), a fluorescently

Transfection of siRNAs and gene knockdown

labeled non‑targeting control chemically modified to prevent

The siGLO RISC-Free Control siRNA was utilized as auptake by the RISC complex. A validated SMARTpool reagent
targeting the human GAPDH (glyceraldehyde-3-phosphatedehydrogenase) gene and an individual siRNA targeting thePPIB (cyclophilin B) gene (siGENOME Cyclophilin B Control
siRNA, Cat #D-001136-01-05) were used as positive controls
for transfection and knockdown efficiency. Untreated cells,cells treated with DharmaFECT 1 alone, and cells
transfected with the siGENOME Non-targeting siRNA Pool(Cat# D-001206-14) or siGENOME RISC-Free Control siRNA
were used as negative controls. The TOX Transfection
Control (Cat #D-001500- 01-05) an RNA oligonucelotide
designed to induce cell death, was used as a positive
control for toxicity. Cells were then incubated for ~ 40 hours
after transfection and fixed with 4% paraformaldehyde.
Separate microwell plates were prepared for eachtransfection plate to determine gene expression andtoxicity. Gene expression levels were determined using thevisual transfection control. The images were acquired on
the ArrayScan VTI HCS Reader using 20x magnification.
Nuclei were stained with 5 μg/mL Hoechst 33342(Molecular Probes, Cat #H1399). The fluorescently tagged
RISC-Free transfection control, observed as punctate spots
and diffuse cytoplasmic fluorescence, suggests that thereis efficient transfection of the siRNA into cells (Figure 1).
To further demonstrate the efficiency of siRNA transfection,
gene silencing was determined by analyzing the functional
knockdown of a positive control SMARTpool reagent orsiRNA targeting either human GAPDH or PPIB. Figure 2 shows
the transfection efficiency and plate variability. Each plateshowed efficient knockdown of PPIB (> 85%) in each wellcontaining the PPIB siRNA and the GAPDH SMARTpool
reagent showed similar transfection efficiency with > 90%
knockdown of GAPDH.

branched DNA assay (Panomics, Fremont, CA). Toxicity wasdetermined using Thermo Scientific™ alamarBlue™ staining(Cat #00‑010).

Figure 1: Individual fluorescent and composite imagesof A549 cells transfected with the fluorescently labeled
siGLO RISC-Free control.
Images were generated by the ArrayScan VTI HCS Reader.
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2High content analysis using targeted RNAi-mediated gene knockdown.

abnormal phenotype: an increase in the 4N population,
consistent with an arrest in mitosis. Knockdown of PLK1 also
induces poptosis as seen in the cell images and the increasein the < 2N population, as well as an increase in the > 4N
population indicating possible necrosis.
Nuclear area analysis
The effect of gene silencing on nuclear area was determinedusing the Cytoplasm to Nucleus BioApplication. An increasein nuclear area is an indication of cell cycle arrest andFigure 2: Efficiency of transfection was determined by functional
knockdown of PPIB or GAPDH expression in the control
wells containing either 100 nM PPIB siRNA or 100 nM GAPDHSMARTpool reagent. Expression levels were determined usinga branched DNA assay and expression was normalized to thePPIB or GAPDH expression level in the lipid-alone negative
control well.

Cell cycle analysis

results from the need to accommodate the increase innuclear material. In Figure 5, the black line represents theaverage nuclear area among cells in control wells and thered and blue lines correspond to two standard deviations
above and below the average. Data points above the redline indicate target silencing that results in larger than
average nuclear area. Consistent with previous studies
demonstrating that mutations in CDC2 result in an arrest

Cells transfected with each of the 80 SMARTpool siRNAin G21, silencing of CDC2 resulted in an increase in nuclear

reagents were analyzed to determine the number ofarea. In addition, silencing of Cyclin A2 (CCNA2) also resulted

cells in each of five cell cycle-related stages (Figure 3)

in an increase in nuclear area. Cyclin A2 (CCNA2) promotes

using the Cell Cycle BioApplication. This BioApplication isboth G1/S and G2/M transitions2. The inset in Figure 5 shows

designed to classify cells and define cell cycle stage byimages (10x magnification) of cells with larger nuclear area.

measuring the nuclear DNA content using the total intensityof a DNA binding dye (Hoechst 33342) for each individualcell. Correspondingly, the cells are classified into one ofA

five classes: less than 2N (apoptotic/damaged), 2N (G1),
2N-4N (S), 4N (G2/M), and > 4N (polyploid or necrotic).

BFigure 3: The Cellomics Cell Cycle BioApplication classifies cells
into their cell cycle phase based on the total nuclear intensity ofa DNA binding dye (Hoechst 33342).

The knockdown of Polo-like Kinase 1 (PLK1) resulted in analtered cell cycle distribution. Data in Figure 4 comparesthe distribution pattern of cells treated with the PLK1
siRNA SMARTpool and the siGENOME Non-targeting Pool.
As expected, the knockdown of PLK1, a key regulatorof mitotic progression in mammalian cells, results in anwww.revvity.com

CFigure 4: Cell cycle analysis of Non-targeting pool and PLK1
siRNAs. A. Cell cycle distribution histogram from the vHCS
Discovery Toolbox of Non-targeting Pool (green) and PLK1
SMARTpool reagent (red) where the Y-axis represents the numberof instances and the X-axis represents the total nuclear intensity.
B. PLK1 and C. Non-targeting Pool: Nuclear images acquired on
the ArrayScan VTI HCS Reader (10x magnification) from the CellCycle BioApplication. Nuclei are shown with either blue or redoverlays where the blue overlays represent cells selected foranalysis and red overlays represent cells rejected from analysis
based on user set criteria.
3High content analysis using targeted RNAi-mediated gene knockdown.

Phospho-ERK (pERK)The Cytoplasm to Nucleus BioApplication can measure theactivation and/or subcellular translocation of individual
proteins from the cytoplasm to the nucleus in single cells.
The BioApplication software is also programmed to analyze
images and calculate the difference between fluorescenceintensities in the nuclear and cytoplasmic regions on a singlecell level and over the population of cells in a well (Figure 7).
Analysis of a downstream phenotypic marker for activationof a signal transduction pathway such as activated
pERK was performed using the Cytoplasm to Nucleus
BioApplication. ERK is phosphorylated in the nucleus upon
Figure 5: Nuclear area acquired on the ArrayScan VTI HCS reader.
The X-axis represents wells transfected with SMARTpool reagentstargeting individual genes and the Y-axis represents the meannuclear area. The black line indicates the average nuclear area ofthe transfection controls and the red and blue lines correspondto two standard deviations above and below the average.
The insets show images of nuclei from cells in which CDC2 orCCNA2 were silenced.

activation of the mitogen-activated protein kinase (MAPK)
pathways. The images in Figure 8 compare cells in which
MCM4 or CDK10 was silenced. MCM4 is required for DNA
replication; therefore, a loss of MCM4 does not result in
nuclear phosphorylation of ERK (non-activated). CDK10 isa CDC2-related kinase that is proposed to play a role inregulating the G2/M phase of the cell cycle and silencingof CDK10 results in an increased level of phospho-ERKAB

Figure 6: Cell Count analysis with representative images acquiredon the ArrayScan VTI HCS Reader (10x magnification) from theCell Cycle BioApplication. Nuclei are shown with either blue or
red overlays where the blue overlays represent cells selected
for analysis and red overlays represent cells rejected fromanalysis based on user set criteria. B. Bar graph representing themean cell number per field with the error bars representing thestandard deviation of the mean of replicate wells.

in the nucleus.

Figure 7: Example of the biology that is measured using theCytoplasm to Nucleus BioApplication.

AB

Cell count analysis
The effect of gene silencing on cell proliferation wasdetermined by analyzing the cell count per field output using
the Cell Cycle and Cytoplasm to Nucleus BioApplications.
Figure 6 compares the representative phenotypes obtainedby silencing of CCNT2, a component of positive transcription
elongation factor b (P-TEFb) and MNAT1, a component ofCDK-activating kinase (CAK). CAK is a multisubunit proteinthat activates cyclin-dependent kinases and functions incell cycle progression, basal transcription and DNA repair3.
Silencing of CCNT2 results in increased cell count while

Figure 8: A. Composite images (10x magnification) acquired on
the ArrayScan VTI HCS Reader from the Cytoplasm to Nucleus
BioApplication demonstrate a lack of pERK in the nucleus ofcells in which MCM4 is silenced and an increased activatednuclear pERK in cells in which CDK10 is silenced. B. Bar graphrepresenting the mean difference between the cytoplasm andnuclear (CytoNucDiff) pERK with the error bars representing thestandard deviation of the mean of replicate wells.

silencing of MNAT1 results in decreased cell count.

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4High content analysis using targeted RNAi-mediated gene knockdown.

Conclusion

References

The results of this study demonstrate compatibility of

1. G.R. Stark, W.R. Taylor, Analyzing the G2/M checkpoint.
Methods Mol. Biol. 280, 51-82(2004).

SMARTpool siRNA reagents with high-content imaging
and anaylsis, and reveal the power of using high content
screening to assess multiple phenotypic changes due to

2. M. Pagano, R. Pepperkok, Cyclin A is required at two points
in the human cell cycle. EMBO J. 11, 961-971 (1992).

specific RNAi-mediated gene silencing. Using highly specificand functional SMARTpool siRNA reagents such as thehuman siRNA library for cell cycle regulation, we show that

3. E.A. Nigg, Cyclin-dependent kinase 7: at the cross-roads ofcell cycle status and phenotypic changes in cell populations
can be easily assayed using HCS instrumentation andtranscription, DNA repair and cell cycle control?
Curr. Opin. Cell Biol. 8, 312-317 (1996).

software. Silencing of specific cell cycle genes (for example,PLK1) resulted in expected phenotypic changes anddemonstrates that the integration of rationally designed
siRNA libraries and the HCS platform offers the means for
rapid and reliable determinations of gene function and for
unprecedented high-throughput strategies.

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