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Dictyostelium discoideum
Microarray analysis

1. Principle
2. RNA extraction
3. Spiking of internal mRNA controls
4. cDNA generation and fluorescent labelling
5. Post-processing of Corning UltraGAPS arrays
6. Hybridisation
7. Washing
8. Scanning of microarrays
9. Signal Quantification

1. Principle
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Figure 1: Principle of DNA microarray analysis
The targets for microarray analysis are two pools of fluorescently labelled cDNAs derived from mRNA of control and experiment cells.
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2. RNA extraction
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Total RNA is extracted from D. discoideum cultures with the Qiagen RNeasy Midi/Mini Kit according to the "Protocol for Isolation of Cytoplasmic RNA from Animal Cells" with the modification of washing the cells twice with water after harvesting to remove medium. Also make sure not to use too many cells, which will result in an incomplete lysis and clogging of the column. After extraction, the RNA concentration is determined by measuring the OD260 (should be > 500 µg/ml) and the RNA is examined on a gel (should give two bands with sizes of 4.1 and 1.9 kb for 26S and 18S rRNA, respectively).
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3. Spiking of internal mRNA controls
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Quality control is an important issue of DNA microarray analysis. Therefore we use the SpotReport Validation Kit, that consists of ten internal mRNA controls from Arabidopsis thaliana genes that are added (spiked) to the D. discoideum RNA prior to cDNA generation and labelling. These mRNAs are provided in a Spikemix with different known amounts of each mRNA. Two different mixes are used for the two labelling reactions (Cy3 and Cy5) of one microarray experiment.

  • Add 1 volume of Spikemix to 1 volume of D. discoideum total RNA (e.g. 10 µl of Spikemix 4A to 20 µg of RNA for Cy3 labelling, 10 µl of Spikemix 4B to 20 µg of RNA for Cy5 labelling).
  • Precipitate the RNA mixes by adding 0.1 volumes 3 M sodium acetate pH 4.8 and 2.5 volumes 100 % Ethanol.
  • Store at -20 °C for 2 hours and centrifuge in a tabletop centrifuge at maximum speed for 30 min.
  • Remove Ethanol by aspiration and wash with 70 % Ethanol.
  • Centrifuge 15 min at maximum speed, aspirate and dry.
  • Dissolve in 12 µl of RNase-free water.
  • Proceed with cDNA generation and labelling.
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4. cDNA generation and fluorescent labelling
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As shown in Figure 1 microarray analysis is performed as a two-channel experiment, where RNA from an experimental condition is compared with RNA from the control. Thus these two RNA-pools have to be labelled with two fluorescent dyes (Cy3 and Cy5). So for each microarray experiment two cDNA-pools have to generated and labelled.
The reverse transcription of mRNA to cDNA is done according to the protocol of the Stratagene Fair Play Kit with the following modifications:

  • Use 0,8 µl of StrataScript and 0,4 µl of RNase Block.
  • Keep all used reagent tubes until the microarray has been scanned, so the LOT number of reagents can be noted if necessary.
  • When dissolving Cy3 or Cy5 in a new vial, label the vial with the date and the amount of dye [µl] remaining after use. Note the LOT Number of the dye package used.
  • During "Dye coupled cDNA Purification" steps 21 ff do not pass the eluate over the column three times, but add 50 µl of 10 mM TrisCl pH 8.5 followed by centrifugation and repeat this step.
  • Determine the cDNA concentration by measuring the absorbance of the eluate at 260 nm in a sterile cuvette. Concentration should be ~ 5 ng/µl.
  • Combine the Cy3 and Cy5 labelled cDNA and ethanol-precipitate as above (chapter 3).
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5. Post-processing of Corning UltraGAPS arrays
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First the microarrays are baked to covalently attach the DNA to the GAPS coating by placing them in an oven at 80 °C for 2 hours, then they are pre-hybridised.
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Pre-hybridisation of Corning UltraGAPS Arrays
Depending on how many arrays are to be pre-hybridised, two different volumes of pre-hybridisation solution are recommended.

A: 90 ml hybridization solution for 5 arrays
B: 400 ml hybridization solution for up to 25 arrays

  A	         B		
20,7 ml    92 ml    water	
22,5 ml   100 ml    20x SSC             
45,0 ml   200 ml    Formamid            
900  µl     4 ml    10 % SDS            
900  µl     4 ml    BSA [10 mg/ml]      

g 5x
g 50 %
g 0,1 %
g 0,1 mg/ml
The washing should be done array-wise and the centrifugation should be performed immediately after the last washing step.
  1. Pre-heat the hybridization solution
  2. Incubate the arrays for 45 min at 42°C
  3. Rinse each array for 15 sec with water
  4. Dip array in isopropanol
  5. Dry arrays by centrifugation at 235xg (1000 upm) for 2 min
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6. Hybridisation
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The microarrays have a barcode to identify each slide and an array area. The array is 18x18 mm in size and was marked with a diamond-pen. For hybridisation we use Corning Hybridisation Chambers (prod.-no. 2551).

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Figure 2: Microarray with barcode and probe area. dummy
Pre-heat a water bath to 37 °C and a heat block to 80 °C

Buffers and solutions:
Hybridisation solution (50 µl)
48µlHybridisation buffer
1µlFish sperm DNA [10 mg/ml]
1µlOligo dA (18 mer, 100 µM)
mix well
 
Hybridisation buffer
1.2MPhosphate buffer pH 6.8
2.0mMEDTA
50.0%Formamide
1.0%Na-Laurylsarcosinate
0.2%SDS
4xDenhardt's reagent
 
100 x Denhardt's reagent
2%Ficoll 400
2%Polyvinylpyrrolidone
2%bovine serum albumin
 
20 x SSC
3.0MNaCl
0.3MNa-citrate
 
1.2 M Phosphate buffer pH 6.8
2vol.1.2 M Na2HPO4
1vol.1.2 M NaH2PO4
 

Procedure:
  • Pipette 10 µl of 3xSSC into the two holes of the Corning hybridisation chamber.
  • Dissolve the precipitated targets in 65 µl of hybridisation solution.
  • Incubate the target solution 10 min at 80 °C.
  • Centrifuge the targets to collect all vapour and pipette the 65 µl of target onto the end of the microarray.
  • Place the cover slip onto the microarray by letting it touch the side of the array first, where the target solution is and then slowly lowering it down until it covers the array area. The target solution should cover the entire array area and there shouldn't be any air bubbles present.
  • Place the slide in the hybridisation chamber, close the chamber and submerge it in the water-bath over night.
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7. Washing
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After hybridisation the microarray is washed to remove unbound target. During washing the transitions from the baths should be performed swiftly, so the microarray does not dry before processing is finished.
  • Remove the microarray from the hybridisation chamber and plunge it into 2X SSC 0,1% SDS until cover slip glides off.
  • Shake in 2X SSC 0,1% SDS for 5 min
  • Shake in 0,1X SSC 0,1% SDS for 5 min
  • Shake in 0,1X SSC for 5 sec
  • Shake in 0,1X SSC for 5 sec
  • Shake in 0,1X SSC for 5 sec
  • Shake in 0,1X SSC for 5 sec
  • Shake in 0,1X SSC for 5 sec
  • Shake in 0,01X SSC for 5 sec
  • Dry arrays by centrifugation at 235xg (1000 rpm) for 5 min.
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8. Scanning of microarrays
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The fluorescent labelled cDNA targets bound to the spots are detected by the ScanArray 4000XL confocal laser scanner. The microarray is scanned for Cy3 and Cy5 successively with a resolution down to 5 µm/pixel. The fluorescent dyes are excited by laser-light of pertinent wavelength and emission is detected by a photo-multiplier. To obtain images well suited for signal quantification image, brightness has to be adjusted by setting the laser-power (photo-multiplier power should always be set at 70 to 80 %). Signals should be as bright as possible, but spots must not be saturated (indicated by white colouring). It might be necessary to scan at two different laser-power settings. One setting where most spots give bright signals, but a few like the some of the positive controls are saturated, and another setting where no saturation is seen, but most spots give weak signals.

  • Switch on the ScanArray scanner and login to the computer (Biomed060 or Biomed061).
  • Start ScanArray Express and switch on lasers 1 and 3 by clicking on the blue buttons.
  • Press Scan, choose Scan type: Run a protocol group and select a protocol by clicking on the bar. Each user should have his or her own protocol group, as it contains an image-auto-save protocol, which will save all images into the users folder.
  • Press Start.
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9. Signal Quantification
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The spot and background intensities of the scanned images are quantified also quantified by SanArray Express.

Before quantifying select the control image via File: Set Control Image!

  • Press Quantitate, select Quantitation type: Run a quantitation protocol and select the appropiate quantitation protocol by clicking on the bar. Because the positions of the sub arrays of the microarrays vary from batch to batch, a different quantitation protocol for each batch exists.
  • Check the alignment of the sub arrays by pressing Adjust Template and Register Images. If necessary move or rotate all or individual sub arrays by dragging with the mouse.
  • Press OK.
  • Press Start.
  • After quantitation the result has to be checked for miss-alignments. Individual spots can be moved and resized by dragging with the mouse and their status (Good, Bad, etc.) may be changed by right-clicking. The distribution plot may help spot irregularities when Footprint, Diameter, Median background or Background Standarddeviation are plotted.
  • Save in Array Informatics via File: Save in Array Informatics.
  • Save CSV and GPR file by selecting the spreadsheet tab and choosing File: Save As.
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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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