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February 09, 2016

Using the Cytation™ 5 Cell Imaging Multi-Mode Reader to Image Printed RNAi Libraries from Persomics

Authors: Paul Held, PhD, BioTek Instruments, Inc., Winooski, VT; Neil Emans, PhD, Persomics USA, Waltham, MA

Personomics

 

RNAi knockdown experiments offer the ability to test the physiologic role of individual gene products through loss of function. Gene silencing through sequence-specific targeting of mRNAs by RNAi has enabled genome-wide functional screens in cultured cells. These screens have resulted in the identification of new cellular pathways and potential drug targets. Typically these cell based screens are performed in 96- or 384-well microplates. Due to the enormous number of genes, hundreds of plates and copious amounts of reagents are required to perform a genome wide screen with redundancy to provide adequate statistics. To address this issue, Persomics has developed a technology that miniaturizes the assay such that the same screen can be performed with only a handful of specialized plates.

Introduction

RNA interference (RNAi) is a biological process in which RNA molecules inhibit gene expression, typically by causing the destruction of specific mRNA molecules. Andrew Fire and Craig C. Mello were awarded the Nobel Prize in Physiology or Medicine in 2006 for their work on RNA interference in C. elegans [1]. In mammalian genomes, two types of small ribonucleic acid molecules, microRNA (miRNA) and small interfering RNA (siRNA), are involved with RNA interference. miRNA molecules are small single strand RNA molecules that base pair with complementary sequences of specific mRNA molecules to form double stranded hairpin loops. Ounce bound, specific messenger RNA (mRNA) molecules generally decrease their activity through targeted cleavage of the mRNA, destabilization of the mRNA by shortening of the Poly(A) tail, or less efficient translation into proteins by ribosomes [2, 3]. siRNA molecules are formed by the cleavage of long double-stranded RNA (dsRNA) molecules into short double stranded fragments of approximately 20 nucleotide siRNA by the enzyme Dicer. Each siRNA is unwound into two single-stranded RNAs, the passenger strand and the guide strand. The passenger strand is degraded and the guide strand is incorporated into the RNA-induced silencing complex (RISC). The guide strand pairs with a complementary sequence in a messenger RNA molecule and induces cleavage by Argonaute, the catalytic component of the RISC complex (Figure 1) [4].

Schematic of RNAi mechanism.

Figure 1. Schematic of RNAi mechanism.

The RNA interference pathway has been exploited in order to investigate the function of genes. Doublestranded RNA is synthesized with a sequence complementary to a gene of interest and introduced into a cell or organism, where it is recognized as exogenous genetic material and activates the RNAi pathway. Using this mechanism, researchers can cause a drastic decrease in the expression of a targeted gene. Studying the effects of this decrease can show the physiological role of the gene product. Since RNAi may not totally abolish expression of the gene, this technique is sometimes referred as a "knockdown", to distinguish it from "knockout" procedures in which expression of a gene is entirely eliminated. RNAi may be used for large-scale screens that systematically shut down each gene in the cell, helping to identify the components required for cellular processes.

Persomic’s Technology

The ImagineArray™ plate is a SBS compliant plate that has no wells. Instead there is a glass slide the same thickness as a class I quartz coverslip fused into the central portion of a molded plastic reservoir plate (Figure 2). Similar to the use of glass bottomed micro-plates, ImagineArray™ plates can be imaged using automated inverted screening systems, or fluorescent microscopes.

ImagineArray™ Plate.

Figure 2. ImagineArray™ Plate. SBS compliant plate that has a glass slide fused into the central portion of the molded plastic reservoir plate.

With siRNA spots (350 μm) printed in discrete addressable locations on the slide, each spot (up to 3200 per plate) is independent from the other microspots. Besides the specific siRNA, each spot contains all the necessary transfection and encapsulation reagents required to introduce the siRNA into those cells adhered to the printed region (Figure 3).

Constituents of a Printed RNAi Microspot.

Figure 3. Constituents of a Printed RNAi Microspot. Microspots positions are identified with a fluorescent label in addition to specific siRNAs, transfection and encapsulation reagents.

The spots are laid out in a matrix with each spot representing a separate RNAi experiment (Figure 4). Specific information regarding each spot can be obtained though multi-color image analysis of the spot and its surrounding regions.

ImagineArray™ Imaging.

Figure 4. ImagineArray™ Imaging. (A) Image montage of a portion of an ImageArray. (B) Enlarged image demonstrating 6 microspots of the boxed portion depicted in figure 4A. (C) Magnified three color image of a single microspot (boxed region of 4B). (D-F) Images shown in figure 4D shown as separate blue, green and red images.

Cytation 5 Imaging Platform

Cytation™ 5 Cell Imaging Multi-Mode Reader is a modular, upgradable multi-mode reader that combines automated digital microscopy and conventional microplate detection. Cytation 5 includes both filterand monochromator-based detection; the microscopy module provides up to 60x magnification in fluorescence, brightfield, color brightfield and phase contrast microscopy. Incubation to 65 °C and plate shaking are standard features. The imaging module uses a turret to hold up to 6 objectives. Excitation and emission wavelengths for fluorescence microscopy are provided using LED light cubes in combination with specific band pass filters and dichroic mirrors. The imaging module holds up to 4 LED cubes. In conjunction with the multimode reader, Gen5 software, which controls reader function, also provides image analysis and data reduction.

Comparison of ImagineArray™ and Multi-Well Screening.

Figure 5. Comparison of ImagineArray™ and Multi-Well Screening. Printed RNAi arrays save considerable amounts of time and reagents as compared to traditional microplate screening techniques. In addition considerable less instrument infrastructure is required.

Performing high throughput siRNA screens using conventional microplate based techniques requires a tremendous investment in time, automation hardware, reagents, plates and other consumables (Figure 5). These costs often prevent individual research labs from performing siRNA screens and have limited both the number and kind of screens conducted to core screening facilities. By using high-density reverse transfection arrays, gene-silencing screens using ImagineArray Plates are faster due to the high number of experiments per array, use less reagents and consumables, and have far fewer handling steps. Here we describe the use of the ImagineArray plate in conjunction with the Cytation 5 Cell Imaging Multi-Mode Reader.

Materials and Methods

An ImagineArray™ siRNA Validation kit was obtained from Persomics. Polyclonal rabbit anti-p65 antibody (cat# SC-109) was purchased from Santa Cruz Biotechnology and Alexa Fluor 488 labeled monoclonal goat ant-rabbit IgG (cat # A-11008) was obtained from Life Technologies. Paraformadehyde (cat# P6148) powder was obtained from Sigma-Aldrich, while Triton X-100 (cat # X198-05) was from Baker.

Cell Culture

HeLa cells were grown in Advanced DMEM supplemented with 10% FBS, 2mM glutamine, Penicillin/ streptomycin Cell cultures were maintained at 37°C, 5% CO2 in a humidified incubator. Cultures were routinely trypsinized (0.05% Trypsin-EDTA) at 80% confluency. For experiments, cells were resuspended in phenol red free DMEM/F12 media supplemented with 10% FBS, 2 mM glutamine, and plated at 1.5 x 106 cells per plate in a total of 15 mL volume. Cells were allowed to grow at 37 °C, 5% CO2 in a humidified incubator for 48 hours.

ImagineArray™ Plate Processing

All liquid handing was performed using a MultiFlo FX with a strip washer module (BioTek Instruments). The Multi-Flo FX has four separate dispense pumps, as well as a 1X8 strip washer dispense and aspiration manifold. Each of the different reagents was added with a separate dispense pump. The ImagineArray was treated as if it was a conventional 8X12 96-well plate by the MultiFlo FX. All reagent additions, wash buffer dispense and aspirations were performed in strip 12. This location is towards one side of the ImagineArray plate and does not impact the microspot array region.

Cells were washed once with 10 mL of PBS (NaCl 137 mM, KCl 2.7 mM, Na2HPO4 10 mM, KH2PO4 7.4 mM) prior to fixation. After which 10 mL of 4% paraformaldehyde (PFA) solution was added with a syringe pump dispenser manifold. PFA solution was made fresh by dissolving 4 g of powder into 100 mL of PBS warmed to 60 °C. Cells were fixed for 10 minutes at room temperature followed by 1 wash of 10 mL using PBS. Cells were then permeablized for 10 minutes using 10 mL of PBS containing 0.1% Triton X-100, added with the second syringe pump. Using the primary peripump dispenser, 10 mL of rabbit polyclonal antibody (1:1000) dilution in PBS, 5% Goat serum) specific for N-kappa-B p65 protein encoded by RELA was added and the plate incubated at room temperature for 60 minutes. The plate was washed five times with PBS 10 mL with a 2 minute soak at room temperature between each cycle. After washing, the secondary Alexa Fluor 488 labeled fluorescent Goat anti-rabbit IgG antibody (1:2000 dilution in PBS, 5% Goat serum) was added and allowed to incubate for 30 minutes at room temperature followed by five 10 mL washes with PBS, again each with a 2 minute soak at room temperature between each cycle. Following antibody binding, nuclei were stained with 10 mL of Hoechst 33342 (10 μM) for 10 minutes at room temperature. Excess stain was removed by washing once with 10 mL of PBS and fresh PBS added to maintain hydration of the samples (Figure 6).

Imaging

ImagineArray™ Plates were imaged using an Cytation™ 5 (BioTek Instruments, Winooski, VT) configured with DAPI, GFP and RFP light cubes. The DAPI light cube is configured with a 377/50 excitation filter and a 447/60 emission filter; the GFP light cube uses a 469/35 excitation filter and a 525/39 emission filter; while the RFP light cube uses a 531/40 excitation and 593/40 emission filters. Exposure settings were manually determined for each color independently prior to imaging, while focus was provided automatically on each location using the DAPI signal. The reader was controlled and data captured and analyzed using Gen5™ Data Analysis Software.

ImagineArray™ Fixation and Staining Process.

Figure 6. ImagineArray™ Fixation and Staining Process.

Image Analysis

Multiple three-color overlaid digital images were electronically stitched using Gen5 software. Object cell counting of the RFP channel was used to identify specific locations of microspots. Subpopulation analysis was used to determine the mean fluorescence intensity of the GFP channel as a means to assess RELA knockdown. Polynuclei determination was made by manual assessment of the number of nuclei in cells within the microspot.

Results

Image analysis of the RFP signal confirms the size of the printed RNAi spot. As shown in Figure 7 the calculated diameter of RFP objects defined using an intensity threshold of 10,000. Cells readily attach and grow on the Persomics plate. HeLa cells seeded in the Persomics plate were monitored kinetically over a period of 24 hours. As shown in Figure 8, these cells will grow not only on the viewing insert, but seem to favor the microspot containing the control siRNA and transfection agents. This suggests that any inhibition of cell growth observed on an experimental microspot is the result of transcript knock-down rather than a generalized cytotoxicity of the microspot in general.

Determined Microspot Size.

Figure 7. Determined Microspot Size. The diameter of 100 microspots was determined using object oriented image analysis of the RFP channel. Data was then expressed as a frequency histogram.

HeLa cell growth on microspot.

Figure 8. HeLa cell growth on microspot. HeLa cells were seeded in a Persomics plate and an individual spot was monitored over 24 hours using bright field imaging. The microspots (white outline) were identified by red fluorescence and subsequent threshold analysis of overlaid image. Time after cell seeding is indicated in each tile along with a magnification scale, which represents 100 μm.

Transcription factor p65 also known as nuclear factor NF-kappa-B p65 subunit is a protein that is encoded by the RELA gene [5]. RELA is a REL-associated protein involved in NF-κB heterodimer formation, nuclear translocation and activation [5]. NF-κB is a pleiotropic transcription factor complex involved in all types of cellular processes, including cellular metabolism, chemotaxis, etc. RELA silencing is detected by immunolabeling of the entire plate. Silenced cells will exhibit diminished fluorescence in the GFP channels as compared to unsilenced control cells. In affected cells cytoplasmic RELA decreases >75% in compared to control siRNA (Figure 9). Measured fluorescence (green) decreased nearly 50% over the region delineated by the array spot as compared to control spots (Figure 10).

Reduction of p65 NF-kappa-B protein subunit expression through RELA silencing

Figure 9. Reduction of p65 NF-kappa-B protein subunit expression through RELA silencing. Overlaid images of discrete ImagineArray™ microspots show qualitative differences in green fluorescent antibody staining of HeLa cells grown over RNAi spots. Top images depict region of the microspot, while lower images have red fluorescence removed for better visualization.

Quantitative Analysis of RELA knockdown.

Figure 10. Quantitative Analysis of RELA knockdown. The mean green fluorescence of 50 microspots from each a control, RELA and INCENP RNAi microspots where determined and compared.

Inner centromere protein is the protein product of the INCENP gene and serves as a regulatory protein in the chromosome passenger complex (CPC). INCENP is the scaffold upon which the CPC assembles [6] The CPC serves as a key regulator of mitosis. It is involved in regulation of the catalytic protein Aurora B kinase. INCENP gene silencing results in a phenotypic change. Because cytokinesis is disrupted, affected cells will often demonstrate multiple nuclei when stained with Hoechst 33342 [7]. As demonstrated in Figure 11, a significant number of HeLa cells cultured over a region containing siRNA targeting INCENP show multiple stained nuclei, while cells not over the silencing spot do not.

Polynuclei induced by INCENP silencing

Figure 11. Polynuclei induced by INCENP silencing. Overlaid images of discrete ImagineArray™ microspots show qualitative differences in the number of nuclei in each cell of HeLa cells grown over RNAi spots. Top images depict region of the microspot, while lower images have red fluorescence removed for better visualization.

A significant increase is observed in the ratio of poly nucleated cells to the total number of nuclei when comparing INCENP silenced cells to those treated with either control or RELA (Figure 12). With control or RELA treated samples only a few cells captured in the midst of mitosis are identified as poly-nucleated. The number of nuclei in INCENP silenced cells represents nearly half the nuclei observed with Hoechst 33342 staining (Figure 12).

Ratio of polynuclei to total nuclei in Negative Control, RELA and INCENP RNAi transfections.

Figure 12. Ratio of polynuclei to total nuclei in Negative Control, RELA and INCENP RNAi transfections. The mean green fluorescence of 50 microspots from each a control, RELA and INCENP RNAi microspots where determined and compared.

Discussion

Persomics' ImagineArray™ Plates enable researchers to silence thousands of genes in a single plate. Each ImagineArray™ Plate contains a pre-printed, ready-torun microarray that can simultaneously silence up to 3200 genes depending on the chosen library. Persomics’ technology encapsulates siRNA and transfection reagent into individual optically addressable spots on a glass substrate optimized for high-content imaging. After cell seeding, no other transfection reagents are required, eliminating a number of the liquid handling steps, as well as the time required to perform those steps, when screening with multi-well microplates. As there are no physical barriers between experiments, cells see the same assay conditions across the entire plate, reducing data heterogeneity. In aggregate, Persomics technology provides the freedom to screen RNAi libraries without automation and costly infrastructure. By streamlining the process, entire siRNA libraries can be screened multiple times using either identical conditions or using multiple different conditions to generate large data sets with superior data quality over screens performed in conventional plates. While Persomics recommends using 10x or 20x imaging to start, higher magnification is possible with longer working distance objectives. You may acquire a series of images to tile together into a single montage in order to visualize the entire array, or set up you imaging system to index to each spot depending on your preference.

The MultiFlo™ FX Multi-Mode Dispenser used for these studies is a modular upgradable reagent dispenser that can have as many as two peri-pump (8 tube dispensers), two syringe pump dispensers and a strip washer. The syringe and washer manifolds can be configured for plate densities from 6- to 384-well. While the low over all plate number used with Persomics technology does not require the use of automated liquid handling, its use reduces the need for manual intervention during the process steps prior to imaging.

The Cytation™ 5 Imaging Multi-Mode Reader is an ideal cost effective platform from which to image RNAi library screens available on the ImagineArray™ plate. Cytation 5 is a modular, upgradable multi-mode reader that combines automated digital microscopy and conventional microplate detection. Cytation 5 includes both filter- and monochromator-based detection; the microscopy module provides up to 60x magnification in fluorescence, brightfield, color brightfield and phase contrast. Incubation to 65 °C and plate shaking are standard features. The imaging module uses a turret to hold up to 6 objectives. Excitatory light and emission wavelengths for fluorescence microscopy are provided using LED light cubes in combination with specific band pass filters and dichroic mirrors. The imaging module holds up to 4 LED cubes. In conjunction with the multi-mode reader, Gen5 software, which controls reader function, also provides image analysis and data reduction.

References

  1. Fire, A., Xu, S., Montgomery, M., Kostas, S., Driver, S., and Mello, C. (1998) Potent and Specific Genetic Interference by Double-stranded RNA in Caenrhabditis elegans, Nature, 391:806-811.
  2. Bartel DP (January 2009). "MicroRNAs: target recognition and regulatory functions". Cell 136 (2): 215-33. doi:10.1016/j. cell.2009.01.002. PMC 3794896. PMID 19167326.
  3. Fabian, MR; Sonenberg, N; Filipowicz, W (2010). "Regulation of mRNA translation and stability by microRNAs". Annual review of biochemistry 79: 351–79. doi:10.1146/annurevbiochem- 060308-103103. PMID 20533884
  4. Bernstein E, Caudy A, Hammond S, Hannon G (2001). "Role for a bidentate ribonuclease in the initiation step of RNA interference". Nature 409 (6818): 363-6. doi:10.1038/35053110 . PMID 11201747.
  5. Nolan GP, Ghosh S, Liou HC, Tempst P, Baltimore D (April 1991). "DNA binding and I kappa B inhibition of the cloned p65 subunit of NF-kappa B, a rel-related polypeptide". Cell 64 (5): 961–9.doi:10.1016/0092- 8674(91)90320-X. PMID 2001591.
  6. Cooke C.A., M.M. Heck, W.C. Earnshaw. 1987. The inner centromere protein (INCENP) antigens: movement from inner centromere to midbody during mitosis. J. Cell Biol.105:2053–2067. 10. 1083/jcb. 105.5.2053
  7. Mackay, A.M., A. M. Ainsztein, D. M. Eckley, and W. C. Earnshaw (1998) A Dominant Mutant of Inner Centromere Protein (INCENP), a Chromosomal Protein, Disrupts Prometaphase Congression and Cytokinesis, J. Biological Chemistry, 140 (5): 991-1002.

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