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Dictyostelium discoideum
Microarray production

1. Introduction
2. Amplification of probes for the microarray
3. Amplification of controls for the microarray
4. Designing the microarray
5. Creating an import file for the MicroGrid II
6. Documentation of microarrays produced and used
7. Printing of microarrays


1. Introduction
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The Dictyostelium discoideum DNA microarray consists of an array of DNA probes called spots. These spots are partial gene sequences or controls. The partial gene sequences for D. discoideum are described in the probe files ddmicro.xls (450 PCR products of known sequence and an average length of 509 bp) and EN-list.xls (~ 5400 plasmid-inserts of ESTs amplified by PCR using a universal primer pair. Controls are selected partial gene sequences from D. discoideum, genomic DNA from D. discoideum, negative controls and the Spot Report-10 Array Validation System.
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2. Amplification of probes for the microarray
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The probes are amplified according to the protocols for the ddmicro- and the EN-list probes. After purification they are resuspended in 10 µl of 25 % DMSO in ddH2O each.
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3. Amplification of controls for the microarray
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The D. discoideum gene controls are amplified by as many PCR reactions as necessary to provide the required volume (N times 10 µl) with a concentration of 100 ng/µl. Additional positive controls are D. discoideum genomic DNA (100 ng/µl) and the SpotReport-10 PCR products. Negative controls are Fish sperm DNA (100 ng/µl), l Bst E III (100 ng/µl), human Cot-1 (10 ng/µl) and SpotReport Poly(dA) (1 ng/µl). N is determined by the desired number of spots per control (see 1.4 last paragraph). For spotting all negative and positive controls are provided in 10 µl of 25 % DMSO in ddH2O (spotting solution). The spotting solution can be used to check for DNA carry-over by spotting directly after the act-A8 partial gene sequence, the strongest positive control.
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4. Designing the microarray
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The D. discoideum microarray generally should contain all available probes and as many controls as needed. The probes are arranged in 96 well microtiter plates and the location of each probe is noted in the probe files (columns "plate" and "well"). The location of a probe determines where it will be located on the microarray and the probe files are used to track all probes during the production process.
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The 96 well format is used for the amplification of the probes, while the 384 well format is used for spotting. Prior to spotting, all probes have to be transferred to 384 well plates according to the 96 to 384 well pipetting scheme. Four 96 well plates are combined to a 384 well plate at a time and the volume per well is 10 µl. A plates file (e.g. A6K PlateSets PinArrays.xls) is created to keep a record of the 384 well plates and to determine how many pin spots are needed. Pin spots are the spots within a sub array. The number of pin spots is limited to 20 * 20 = 400 (allowing for 400 x 16 sub arrays x 3 arrays = 19200 spots per slide). The position of the pin spots can be chosen freely and numbers can even be skipped, which makes it possible to include incomplete 384 well plates. As the pin tool prints 16 sub arrays simultaneously a pin spot refers to 16 wells in a 384 well plate and it also means that all sub arrays are printed with the same pin array. The pin array is the printing scheme for the sub arrays. It can be conveniently created with a spreadsheet software (e.g. MS Excel; A6K PlateSets PinArrays.xls) and used for documentation and spotting, when converted to CSV (column separated values) format (also see The MicroGrid II User manual).
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The controls are a small group of DNA solutions that were selected individually and which can be changed easily. The controls are directly pipetted into a 384 well plate (the format used for spotting) and a record is kept in a controls file (A6K Controls.xls). Usually the controls are printed in replicates onto the microarray, so they appear in four or even all sub arrays. Therefore the controls have to be provided in as many wells of the 384 well plate, as the number of sub arrays they are printed in. Thus larger volumes of controls (e.g. 160 µl for 16 wells/sub arrays) are needed.
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5. Creating an import file for the MicroGrid II
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As all probes are rearranged during the spotting process an import file has to be provided for the MicroGrid II Clone Tracking Wizard, which creates an output file (GAL file) used by ScanArrayExpress, the spot quantification software.
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The input file can be created using the Input File Creator tool, which uses the ddmicro.xls, EN-list.xls and controls.xls files.
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The MicroGrid II Clone Tracking Wizard can then be used to create the output file. Creation of the output file is independent of the spotting process and can be done any time.
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6. Documentation of microarrays produced and used
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The production and use of all microarrays has to be tracked. In the slides files (e.g. slides 020403.xls) the printing conditions, post-processing steps and use is recorded for future use.
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7. Printing of microarrays
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The printing is described for the microarray A6K. The D. discoideum microarray A6K is made up of three pin arrays. As the same 384 well plate is used to spot the controls in all three pin arrays, the A6K array has to be printed in three runs. The 384 well plates used for each of the three runs are described in the plate set (A6K PlateSets PinArrays.xls).
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Each spotting run is documented with a Spotting Check List; before starting a run all settings have to be checked with this list. Prior to spotting the MicroGrid II has to be vacuum cleaned and the ddH2O of the MWS (multi wash station) exchanged. Start the computer and log on locally. This is important, because the regional settings of Windows will change from English (UK) to German otherwise. The humidifier and afterwards the main unit, and cooling device need to be switched on and the TAS Application Suite started. A microarraying run can easily be set up, by modifying a previous run (e.g. a5826_split.grid). In this case only the parameters concerning the new microarray design have to be entered, while the preferences remain. For detailed information refer to The MicroGrid II User manual. For the printing run the 384 well plates are briefly centrifuged (e.g. 250 xg, 1 min) without the heavy lids, then they are covered with the lids and are placed into the BioBank.
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Sigma Poly-L-Lysine slides are used for pre-spotting and UltraGAPS are used for the microarrays. They are placed inverted onto the first tray, to avoid contact of the pins with the labelling area, and their number is calculated by the MGII software. All slides are dusted with the antistatic brush or vacuum cleaned. The slides are placed on the trays (Note that the slides are printed from left to right in the first and third row, but from right to left in the second row) and aligned with the front left corner of each holder. The barcodes of the slides have to be noted for documentation in the slides file (e.g. A6K Run001 slides.xls). Empty spaces on a used tray have to be covered with dummy-slides, before the vacuum can be applied to hold the slides. Now the slides are ready for printing.
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After printing and before post-processing the spots have to dry on the slides.
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September 12, 2013
Institute of Biochemistry I, Cologne
Suggestions and wishes: Gudrun Konertz
Voice: +49 221 4786930
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