Production of siRNA and cDNA-Transfected Cell Arrays on Noncoated Chambered Coverglass for High-Content Screening Microscopy in Living Cells
In this chapter, we provide a protocol for the production of transfected cell arrays in living mammalian cells on noncoated chambered coverglass for the systematic functional analyses of human genes by high-content screening microscopy. This method should facilitate drug target validation by small-interfering RNAs.
Key WordsCell arrays genomics high-content screening microscopy small-interfering RNAs siRNA
The information available through sequencing of several genomes together with high-throughput techniques such as protein analysis by mass spectrometry or expression and transcription profiling by protein or DNA microarrays has the potential to help analyze the complexity of biological processes on a comprehensive scale (seeChapter 2, Chapter 4, Chapter 5, Volume 1). An elegant high-throughput method allowing parallel analysis of gene function in intact living cells has been introduced recently by Ziauddin and Sabatini (1). In this method, plasmid DNAs or smallinterfering RNAs (siRNAs) (2) are printed together with the appropriate transfection reagents in a gelatin matrix at defined locations on glass slides. Overlaying these arrays with tissue culture cells results in clusters of living cells transfected with siRNAs or expressing the respective cDNAs at each location. In combination with automated fluorescence scanning microscopy and image processing (3,4), this method allows the rapid analysis of gene function on a large scale in intact cells.
siRNA oligonucleotides (QIAGEN, Valencia, CA).
Cy3-labeled DNA marker oligomer (BioSpring, Frankfurt, Germany).
384-Well plates (Nalge Nunc International, Rochester, NY).
Lab-Tek chambered coverglass (cat. no. 155361, Nalge Nunc International).
Transfection reagent Effectene (QIAGEN, Hilden, Germany) or LipofectAMINE 2000 (Invitrogen, Carlsbad, CA).
Sucrose (USB, Cleveland, OH).
Gelatin (cat. no. G-9391, Sigma, St. Louis, MO).
3.1 Sources of Tagged cDNAs and siRNAs and Preparation of Transfection Solutions
3.1.1 Sources of Tagged cDNAs and siRNAs
3.1.2 Preparation of Transfection Solutions
The siRNA (plasmid cDNA) gelatin transfection solutions are prepared in 384-well plates (Nalge Nunc International). As transfection reagent, we use Effectene (QIAGEN) or LipofectAMINE 2000 (Invitrogen), giving optimal transfection efficiencies for both siRNAs and plasmid cDNAs in MCF7, HeLa, COS7L, or human embryonic kidney (HEK)293 cells. The presence of sucrose in the spotting solution facilitates the storage of the dried arrays without loss in transfection efficiencies. Additionally, it is essential for the successful transfer of the siRNA (cDNA)-gelatin transfection solutions to uncoated substrates during the spotting procedure. The presence of fibronectin in the gelatin solution increases cell adherence, resulting in a reduced migration of transfected cells away from the spot region. To retrieve the spot regions and to highlight successfully transfected cells of siRNA experiments, a Cy3-labeled DNA oligonucleotide is used as cotransfection marker. In this case, 0.5 µL of a 40 µM marker solution is included in step 1 of the protocol, resulting in a total oligonucleotide volume of 1.5 µL (1 µL of siRNA plus 0.5 µL of Cy3-labeled oligonucleotide). With this protocol, it is possible to cotransfect plasmid cDNA and siRNA. In this case, 1 µL of siRNA plus 1 µL of plasmid cDNA are added in step 1, resulting in a total volume of 2 µL.
Add 1 µL of the respective siRNA stock solution (20 µM in RNA dilution buffer as supplied by the manufacturer) to each well. For plasmid transfections, 1 µL of plasmid DNA at a stock concentration of 500 ng/µL is added.
Add 7.5 µL EC buffer (part of the Effectene transfection kit, QIAGEN) containing 0.2 M sucrose and mix thoroughly by pipeting three times up and down. The EC buffer can be replaced by water, also containing 0.2 M sucrose for LipofectAMINE 2000 (Invitrogen).
Incubate the mixture for 10 min at room temperature.
Add 4.5 µL of Effectene transfection reagent (QIAGEN) or 3.5 µL of LipofectAMINE 2000 (Invitrogen).
Incubate for 10 min at room temperature.
Add 7.25 µL of 0.08% gelatin (G-9391, Sigma) containing 3.5 × 10−4% fibronectin (Sigma).
Spin down the 384-well plate for 1 min at 216g. The solution is now ready for the spotting process.
3.2 Spotting the Transfection Solution on Uncoated Lab-Tek Dishes
As a substrate, we use untreated chambered coverglass dishes, allowing live cell imaging and easy-to-perform immunostaining. We use a ChipWriter Compact robot (Bio-Rad, Hercules, CA) equipped with PTS 600 (Point Technologies) solid pins. The distance between the spots is 1125 µm, resulting in spot diameters of approx 450 µm. The spotted solutions on the Lab-Tek chamber are dried at room temperature for at least 12 h after printing before cells are plated. The solutions of one 384-well plate (seeSubheading 3.1.) are sufficient to spot at least 50 identical Lab-Tek chambers, which can be stored for later use in a dry environment for several months without obvious loss in transfection efficiencies. The outer dimensions of a Lab-Tek chamber are 57.2 × 25.6 mm (length × width). Of this area we use, 34.875 mm in length and 12.375 mm in width to spot 384 samples. Spotting 384 samples on 48 Lab-Tek chambers, by using eight solid pins in the x-direction mode typically lasts 6 h.
We adjust the temperature of the 384-well plate with an in-house-built water-cooled plate to 12°C.
The dwell time the spotter pins remain in the source plate is set to 0.5 s.
The dwell time the spotter pins remain on the Lab-Tek chamber is set to 0.1 s.
- 4.The washing procedures between the spotting of distinct samples are set as follows:
Wash container: 10 s.
Sonication container: 10 s.
Vacuum drying: 10 s.
3.3 Plating of Cells on Lab-Tek Dishes With Dried Transfection Solution
The density of the cells plated on the dried spots of a Lab-Tek chamber is always a compromise between the improved statistics that can be achieved with high cell densities and the quality of the microscopic analyses that is best at low cell densities.
Typically, we plate 1.25 × 105 actively growing HeLa, MCF7, COS7L, or HEK293 cells in 2.5 mL of culture medium (Dulbecco’s modified Eagle’s medium containing 10% heat-inactivated fetal calf serum, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/mL streptomycin) on the dried spots of one Lab-Tek culture dish. This process results in approx 100 to 200 cells residing on one spot.
The incubation time (at 37°C and 5% CO2) for the successful expression of plasmid cDNAs varies between 12 and 24 h.
The incubation time for RNA interference (RNAi) experiments varies between 20 to 50 h and strongly depends on the stability of the proteins targeted by the siRNAs spotted. For long-term experiments lasting several days, e.g., RNAi experiments targeting stable proteins, the cell density needs to be lowered compared with the density typically used (seestep 1), because cells may stop growing because of contact inhibition at later time-points of the experiment (e.g., 60 h), which makes experiments addressing cell cycle- or signal transduction-related questions difficult to interpret.
3.4 Preparation of Samples for Functional Analysis
For functional analysis involving high-content screening microscopy, we frequently use immunofluorescence (6) to monitor molecule-specific morphological and biochemical parameters. The immunostaining procedure in Lab-Tek chambered glass cover slips is very effective and cheap because it can be performed with the same antibody for hundreds of target genes in parallel.
For the staining of Lab-Tek chambers, we apply 250 µL of the corresponding-antibody by carefully distributing the fluid over the spotted area.
Incubate for 10 min with the lid closed to avoid rapid evaporation.
Wash two times with 2 mL of phosphate-buffered saline (PBS) (30 min each).
Stain cell nuclei with 0.2 µg/mL Hoechst B2261 (Sigma). The strong nuclear staining achieved in this way facilitates automated focusing during image acquisition (3) (seeSubheading 3.5.).
The stained samples are stored at 4°C either embedded in Mowiol or in PBS solution containing 0.1% azide after a brief poststaining fixation of the samples with paraformaldehyde for 2 min.
3.5 Analysis of Samples by High-Content Screening
In principle, images of the cells on the spots can be acquired with any commercially available inverted microscope. We use a ScanR (Olympus Biosystems, Munich, Germany; ) scanning microscope, with automated focus, allowing time-lapse data acquisition. The microscope is equipped with standard filters to detect DAPI, GFP, and Cy3. A 10 × /0.4 air or a 40 × /0.95 air PlanApo objective (Olympus) is used for image acquisition.
A key step of the whole image acquisition process is to find the first spot of the array. We use the fluorescent signal of the cotransfected Cy3 DNA oligonucleotide. In addition, we mark the first spot manually with a thin and water-resistant black marker pen on the opposite side of the coverglass where the spots are located, before cell seeding.
Assign the spot-to-spot distance, the number of samples, and the array dimensions (number of samples in x-direction and number of samples in y-direction).
For image processing the images are background corrected by subtracting the average pixel value in a blank region of the image. The locations of the cells are determined by Hoechst nuclear stain. A possible fluorescence statistics can be based on the mean fluorescence values in a dilated region around the nucleus (including or not including nucleus, depending on the assay). Average values obtained should be normalized to the siRNA control (1.0) (nonsilencing) spotted on the same slide.
The spotting robot used has to be able to pass the walls of the Lab-Tek chamber.
To achieve the required accuracy of the spot-to-spot distance, high-resolution spotting robots are necessary.
- 6.Pepperkok, R., Girod, A., Simpson, J. C., and Rietdorf, J. (2000) Imunofluorescence microscopy, in Shepherd, P. and Dean, C., eds., Monoclonal Antibodies: A Practical Approach. Oxford University Press, New York, pp. 355–370.Google Scholar