Protocol

Analysis of siRNA Knockdown of Cell-Cycle Control Genes in G₁/S and G₂/M Cell-Cycle Phase Marker Cell Lines Using Multiplexed High-Content Analysis

This protocol was adapted from "High-Content and High-Throughput Screening," Chapter 13, in Cell Imaging (ed. Stephens). Scion Publishing Ltd., Oxfordshire, UK, 2006.

INTRODUCTION

RNA interference using siRNA libraries is a powerful technology for elucidating gene function by downregulating gene expression at the post-transcriptional level. Phenotypic changes associated with siRNA knockdown can be monitored using cell lines expressing fluorescent reporter proteins. Test siRNAs are transiently transfected into the reporter cell line and behavior of the fluorescent reporter probe is monitored using a high-content imaging system. A range of cell features and additional fluorescent probes can be monitored to assess the effects of knockdown. This article provides a method of screening an siRNA cell-cycle array using cell lines that report cell-cycle phase. The subcellular distribution and intensity of the fluorescent phase reporter changes in a cell-cycle-dependent manner. The phase marker assay is multiplexed with an assay for bromodeoxyuridine (BrdU) incorporation, which identifies cells that have undergone DNA replication. When a G₂/M cell-cycle phase reporter cell line is multiplexed with BrdU incorporation, information from both assays enables assignment of each cell to one of five distinct phases of the cell cycle (G₁, S, G₂, prophase, or mitosis).

RELATED INFORMATION

Protocols for Preparation of Cells in 96-Well Plates for siRNA Transfection and High-Content Analysis and Optimization of siRNA Knockdown in EGFP-Expressing Cell Lines Using High-Content Analysis are also available.

MATERIALS

Reagents

Cell Proliferation Fluorescence Assay kit (with BrdU labeling reagent; GE Healthcare)

Culture medium

DharmaFECT 3 transfection reagent (Dharmacon)

DharmaFECT cell-culture reagent (Dharmacon)

Formaldehyde solution, neutral buffered (4%; Sigma)

G₁/S and/or G₂/M cell-cycle phase marker cells (GE Healthcare), suspended at 5000 cells/100 µL in antibiotic-free medium with 10% FBS (see Preparation of Cells in 96-Well Plates for siRNA Transfection and High-Content Analysis)

Hoechst 33342 (2 µM in 1X PBS; Sigma)

Phosphate-buffered saline (PBS) (1X; sterile, calcium- and magnesium-free)

siRNA plate (cell-cycle siARRAY RTF siRNA 96-well imaging plate; GE Healthcare/Dharmacon)

Triton X-100 (Sigma)

Equipment

Incubator, tissue culture (37°C, 5% CO₂, humidified)

Microscope, automated fluorescence, with emission and excitation filters for Hoechst, EGFP, and Cy5, and image analysis software (e.g., IN Cell Analyzer 1000 or IN Cell Analyzer 3000; GE Healthcare)

METHOD

1. Equilibrate the siARRAY plate to room temperature.

2. For each well to be transfected, combine 0.125 µL of DharmaFECT 3 with 24.875 µL of DharmaFECT cell-culture reagent and add 25 µL to each well of the siARRAY plate. Incubate for 20-90 min at room temperature to rehydrate the siRNAs.

3. Aliquot 100 µL of cell suspension into each well containing rehydrated siRNAs, and incubate for 24-48 h in a tissue culture incubator.

4. Dilute BrdU labeling reagent (from the Cell Proliferation Fluorescence Assay kit) 1:250 with tissue culture medium, add 100 µL per well, and incubate for 1 h at 37°C.

5. Remove the medium and wash the cells with 1X PBS by gentle aspiration.

6. Fix cells with 100 µL of 2% formaldehyde/0.1% Triton X-100 for 30 min at room temperature or overnight at 4°C.

7. Stain cells for BrdU incorporation using the detection reagents supplied in the Cell Proliferation Fluorescence Assay kit.

8. Remove formaldehyde, wash cells with 1X PBS, and stain nuclei with 100 µL of 2 µM Hoechst 33342 in 1X PBS.

9. Image the cells using excitation and emission filters for Hoechst, EGFP, and Cy5.

10. Analyze the images for cell number, cell-cycle distribution, BrdU incorporation, and cellular morphology parameters using the appropriate image analysis software (e.g., Fig. 1 ).

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Figure 1. Analysis results from an siRNA study using a G2/M cell-cycle phase reporter cell line. Images from wells treated singly (1-4) or in combination (pool) with four siRNAs directed against different regions of the mRNA for polo-like kinase, which is a regulator of cell-cycle progression during mitosis. An EGFP fusion protein (green) reports cell-cycle phase; nuclei are stained with Hoechst (blue), and nuclei that have incorporated BrdU are stained with Cy5 (red). For each image, the corresponding analysis results are displayed as a pie chart that shows the proportion of cells in mitosis (M), G1 phase, S phase, G2 phase, and prophase (P). Changes in cell-cycle phase distribution can be assessed relative to the distribution determined from cells in the control well, which received a pool of scrambled siRNAs. (Reprinted with permission, © 2005 Scion Publishing Ltd.).

See Troubleshooting.

TROUBLESHOOTING

Problem: Background is high and/or analysis is unsuccessful.

[Step 10]

Solution: When working with the cell-cycle phase marker cell lines, take care to minimize cell disruption during wash, permeabilization, and antibody incubation steps, and avoid creating vortices when adding reagents to wells. Cells that are dead, dying, dividing, or blocked in mitosis may be weakly adherent and therefore particularly susceptible to disturbance. Selective loss of these cell subpopulations will skew the analysis results. Keep the formaldehyde incubation time (Step 6) between 20 and 30 min to avoid under- or overfixing the cells. To minimize background fluorescence, use the plates, serum, and culture medium from the recommended suppliers (see Preparation of Cells in 96-Well Plates for siRNA Transfection and High-Content Analysis).

Problem: Image quality needs improvement and/or analysis is unsuccessful.

[Step 10]

Solution: Inspect images to ensure that field illumination is even and that features to be quantified are bright and in focus. If not, adjust the instrument and acquisition set-up and reimage the samples. If reacquisition is not possible, try applying noise reduction or shading correction techniques before analysis. Overexposure of images can be as detrimental as underexposure. Check pixel gray-level values of representative cell features to ensure that they fall within the dynamic range of the camera. If not, reimage the sample using a shorter exposure time, decrease the laser power, or add a neutral density filter. Good segmentation of nuclei, cell bodies, and features of interest is also critical for success. When creating a new analysis protocol, assess the analysis results and ensure that the software has identified the targeted population of cells (and is not, for example, mistaking cell debris for legitimate cells). Most analysis software provides visualization tools such as bitmap overlays that allow you to assess the results. When using IN Cell Analyzer 1000 software, if too few or too many objects are detected, try systematically varying the sensitivity settings during object segmentation. For cell identification in particular, try varying the segmentation method as well. When creating a classification protocol, ensure that the image quality of the training data set is excellent and that all objects of interest are well segmented before proceeding to annotation.

ACKNOWLEDGMENTS

The author gratefully acknowledges Nick Thomas and Mike Kenrick for the methods and data described in this article.