A Plate of Chips
Something New on the Menu for Microarrays
To download this tutorial as a PDF, please click here.
Plate Arraying
1. Plate arrays—the challenges:
- Production of high density multiplexed arrays
- Increasing throughput to allow walk-away automation
- Depositing samples onto delicate surfaces
- Avoiding cross contamination
- Maintaining sample stability
- Minimize dust contamination within arrays
Plate Arraying layout on MicroGrid II
2. Plate arrays—details on our solutions:
High density multiplexed arrays
Adapter plates are available for both instruments enabling the user to print arrays into individual wells of
microplates rather than onto slides. The GeneMachines OmniGrid Accent can array into 6 destination plates
whereas the BioRobotics MicroGrid II has three times greater caspacity, holding 16 microplates. The Genomic
Solutions microarray platform has been designed with flexibility as a priority and can accommodate many plate
definitions for this application. A library of definitions for most commonly available microplates is available in
the software allowing simple set up of arrays into the users' choice of plates.
Plate Arraying layout on OmniGrid Accent
In house research has shown it is possible to array and accurately quantitate at least 1000 DNA or protein spots in a single well of a 96 well microplate or 450 features in each well of a 384 well microplate. Arrays can be printed in either a circular or square format to ensure maximum use of well space.
Genomic Solutions provides flexibility in plate configuration enabling researchers to array from 96 wells into 384 wells and vice versa as well as between plates of the same format.
These high density arrays are produced using contact printing with Telechem 946 pins or MicroSpot 2500 or 10K split pins*. These split-pin designs load up to 250nl of sample into the pin by capillary action, covering the end of the pin tip with a thin layer and allow printing by simple surface tension and adhesion. This benefits the researcher by enabling samples to be deposited onto delicate surfaces with minimal contact between pin and substrate.
Due to the viscous nature of proteins researchers may choose to move away from split-pin technology for the plate arraying of proteins. Genomic Solutions can offer these scientists the option of proprietary MicroSpot solid pin technology.
Increasing throughput with walk-away automation
The GeneMachines OmniGrid Accent can hold 3 source plates and 6 target microplates. The BioRobotics MicroGrid II has almost three times the target plate capacity and can hold 24 source plates in a cooled environment. These configurations enable a researcher to improve assay production by up to a factor of fifty, compared to standard slide production methods. Both instruments are compatible with 96 and 384 well microplates.
To enhance walk-away usability further Genomic Solutions has also developed microarray software for plate-based applications. Both instruments have flexible software offering a fully customizable array design feature a simple wizard guides the operator through target plate selection, pin tool configuration and source options. The user is then taken through array design and layout as well as pin washing options. After a summary of the parameters selected the plate-arraying run will begin.
An essential sample tracking function within the software ensures traceability from source plate well to individual spot location within each microplate well by the production of a .gal file.
Depositing samples onto delicate surfaces
Control of spotting speed using Genomic Solutions proprietary Soft Touch feature allows precise and gentle printing onto the surface of the well, particularly useful when arraying onto a coated surface. It improves spot morphology and also reduces pre-spotting enabling higher throughput and ensuring valuable sample is not wasted.
In more detail, when you are moving your array printhead around to spot your pins onto your array substrates, you move it at a particular speed and normally the speed is very fast. Soft Touch allows you to quickly decelerate the pin to a very slow speed just before the point of contact. This gives you much better spot definition and morphology by doing this quick deceleration process just before you touch. Once you have finished the touch, it re-accelerates back to the original speed.
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| A screen shot from BioBio Robotics MicroGrid II Plate Array Software |
Avoiding cross contamination
Thorough pin cleaning is extremely important for successful arraying, especially for protein applications; therefore Genomic Solutions offers pin sonication in addition to multiple wash stations.
Maintaining sample stability
Genomic Solutions arrayers can include source plate cooling. This is especially important for helping to ensure that proteins remain stable prior to transfer to the substrate surface. Both the BioRobotics MicroGrid II and the GeneMachines OmniGrid Accent have humidification as standard to prevent sample evaporation.
Reduce dust contamination within arrays
Both the BioRobotics MicroGrid II and the GeneMachines OmniGrid Accent use HEPAfiltration to minimize dust present within the system, ensuring the printing of clean, high quality arrays.
Protein Arraying
3. Protein arrays—challenges:
Protein microarrays offer the ability to measure protein expression and antibody binding in a high-throughput and miniaturized manner. Over the years, Genomic Solutions has developed instruments to enable automated workflow process of DNA microarray production, hybridization and data analysis. The ability to use these instruments for protein microarrays greatly increases their functionality and streamlines the process of generating data. Unfortunately, there has been a lack of information on the processes involved in using DNA microarray equipment for the protein microarray workflow. Protein microarrays have some unique features that may make DNA microarray instrumentation difficult to transition. Here are some of the common challenges relating to protein arraying:
- Hard to set up protein array parameters
- Concerns on carry-over contamination
- Concerns over protein-protein binding
4. Protein arrays—our solutions to the challenges:
Here are some of our solutions to overcome these challenges:
Optimization of Protein Array Parameters
A major problem with the optimization of protein array printing is the time and cost associated with the set-up and analysis of protein-protein interactions. Optimization using fluorescent dye eliminates the use of costly experimental samples in order to evaluate the optimization process. We recommend using some type of fluorescent substance (e.g. red food dye resuspended in 1X PBS) to allow visual inspection of spot deposition. By scanning slides printed with red food dye in the Cy3 channel it is possible to get a high resolution image of the spots to use for optimizing printing conditions.
Minimizing Carry-over Contamination
Due to the composition and differing viscosity of various spotting solutions used for protein arrays, minimizing the amount of carry-over between spots is a critical step for successful protein arraying. The Genomic Solutions Omnigrid and Microgrid microarraying systems allow for complete control over the wash steps and cycles.
In order to establish a wash protocol that did not have carry-over, each dip into the dye sample was spotted 6 times onto the slides followed by the wash cycle, dipped into 1X PBS without dye and printed for an additional 6 spots. This was repeated three times. By varying the number of cycles through the sonication, wash, and dry cycles it is possible to develop a wash protocol that does not have any carry-over between samples.
The Omnigrid system has the advantage of an on board sonication system to increase the effectiveness of the wash cycles. The Microgrid system has two recirculating wash baths and uses the flood and flush method of cleaning the pins. Additionally, the two recirculating wash baths on the Microgrid can be used as stationary baths, which can be filled with various cleaning agents such as ethanol. Although the 2 systems have different wash setups, wash methods on both systems were shown to be equally effective for cleaning pins.
Due to variations in printing solutions and concentration of proteins, sample specific wash protocols will need to be determined empirically. By varying the times and number of cycles through the wash stations, it is possible to print proteins on nitrocellulose without any visible carry-over.
Protein-Protein Binding
When proper printing and wash protocols have been established, the next steps are printing followed by addition of the solution-phase binder. Protein arrays may have complex binding protocols involving multiple blocking and wash steps. The Genomic Solutions Hybstation is a convenient way to automate complex solution-phase binding procedures.
Standard protein solution-phase binding protocol can be written using the Methods Editor software program of the Hybstation. Blocking, solution-phase binding, and wash steps can be included in the protocol and antibody binding may be carried out overnight. The slides were then allowed to air dry prior to scanning. This basic program can be modified and adjusted easily with the Methods Editor software according to the individual researcher needs.
SUMMARY
In conclusion, there are several challenges to the production of plate arrays. Genomic Solutions can now offer high quality plate arraying as an option or upgrade to the existing BioRobotics MicroGrid and the GeneMachines
OmniGrid Accent. This technology is part of a suite of products from Genomic Solutions designed to assist the researcher in meeting the challenges of achieving a multiplexed approach to DNA and protein microarraying. With this technology advance, plate and protein arraying may finally become a reality to serve the need of more diagnostic applications.
To download this tutorial as a PDF, please click here.
For a demonstration of this new technology or advice on you current microarray applications, please contact us at: arraydesign@genomicsolutions.com
*MicroSpot split pins not available in USA or Canada