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Standard Operating Procedure

S.O.P#

IGF018.00

Title

Hybridisation of labelled cDNA’s with microarrays using the Genomic Solutions Hybridisation stations.

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1.0

BACKGROUND
To measure levels of gene expression, test and control total RNAs are used to produce fluorescently labelled cDNA targets. These are hybridised, using an automated hybridisation station from Genomic Solutions to control the hyb conditions, with PCR amplified DNA probes which have previously been immobilised on Corning GAPS slides. The resulting fluorescent intensities detected on scanning the slides with the appropriate lasers to excite the fluorescent label, provide information on the levels of expression of the particular DNA product in the tissues from which the test and control RNAs were derived.

2.0	SLIDE CASSETTE ASSEMBLY
	2.1	Switch on hybridisation station and refer to SOP026.00 for required maintenance checks if the machine has not been used for some time.
	2.2	Remove the required number of cassettes to accommodate the number of slides being hybridised and separate the black base from the clear cover.
	2.3	Ensure that the clear cover has four small, red O rings on the top edge and place a larger black O-ring around the edge of each of the two slide chambers. Ensure that the assembly is clean and free from dust.
	2.4	Remove the box of slides from the desiccator and place two of the printed slides on to the black base, array uppermost, and note which slide goes into which cassette and in which position. If the slide has a barcoded area then this should be placed nearer the top of the black base, near the raised U-shaped metal piece.
	2.5	Tilt the base back to position the slides at the top right corner of the holder. If only one array slide is required, ensure that a blank has been placed in the other position on the base.
	2.6	Carefully, place the clear cover over the slide/base assembly. Place the U-shaped metal piece at the top edge of the base into the recess on the edge of the cover.
	2.7	While holding this “sandwich” together, insert it into one of the Hybridisation station modules. Use the U-shaped metal piece and the metal pin in the centre of the Plexiglass manifold at the top of the module to position the assembly correctly. Push the slide assembly up so that there is good contact between it and the Plexiglass manifold.
	2.8	Lower the clamp over the slide cassette and locate the locking screw on the correct thread to screw the assembly down and hold in the correct position. Tighten the locking screw until it can no longer be easily turned by hand.
	2.9	Ensure the wash bottles contain enough liquid and are in the following order; Bottle 1, medium stringency wash, Bottle 2, high stringency wash and Bottle 3, post wash buffer.
3.0	MAKING UP HYBRIDISATION SOLUTION
	3.1	Ensure the hot block in the clean room is set to 95°C. The hybridisation solutions should be made up in individual tubes in the wet lab and transferred to the clean room for the final denature before injection into the slide chambers.
	3.2	Remove the appropriately labelled cDNA’s from the freezer and thaw. Cover with foil to protect the label from light during this time.
	3.3	Prepare the hybridisation solution for each slide as below:
			Solution 50:50 ULTRAHyb:2 x SSC PolyA (10mg/ml) Cy3 label Cy5 label Salmon Sperm DNA (10mg/ml) Ultra pure BSA (50mg/ml)	Volume 85ul 10ul 10ul 10ul 5ul 5ul

4.0	LOADING HYBRIDISATION STATIONS
	4.1	On the Hyb Station press the Start a Run option from the main menu. You will be asked where the program you wish to run is stored, choose Floppy Disk from the list of options. The current protocols are stored within the Hyb Station protocols folder in SourceSafe and can be copied from there onto a floppy disc.
	4.2	Press the name of the program that you wish to load to select it – currently the standard protocol used is named 42 low wash. Once selected press Load.
	4.3	A diagram of the six modules on the Hyb station will now be displayed. Touch the modules, into which you previously loaded the full cassettes, these will be highlighted in the display. There is no need to select any blank slide positions you have within the cassettes being used.
	4.4	When all the modules being used have been selected press Start. You will now be asked to check that all the cassettes are in position, and then press Continue. The modules will now start ramping up to the starting temperature of 75°C for the O-ring conditioning.
	4.5	Once the O-ring conditioning has completed the screen will display a window with Probe and Finish buttons for the two slides within the first slide cassette requiring injection of the hybridisation solution. WARNING, the modules do not necessarily become available for injection in numerical order. You MUST read the screen to ensure you will inject the hybridisation solution into the correct position.
	4.6	Place the two microfuge tubes containing the hybridisation solution for the two slide positions displayed in the hot block at 95°C for two minutes.
	4.7	This will open an inlet valve within the system to allow the hybridisation solution to be injected correctly, you should hear a click.
	4.8	Pulse spin the heated hybridisation solution to bring the contents of the tube to the bottom and aspirate using a 200ul Gilson pipette with a Greiner 200ul plugged tip (N.B. do not use normal yellow tips since they do not seal as well and you will experience problems loading the chamber).
	4.9	Insert the tip of the pipette into the probe injection port on the appropriate slide chamber. VERY slowly depress the plunger and inject the fluid into the chamber. Care should be taken to maintain an even pressure to avoid bubble formation on the slide. Continue until the fluid fills the chamber.
	4.10	Remove the pipette tip and insert a plastic probe plug into the port. On the screen press Finish for the first slide position, and then repeat the process for the second slide in that module unless it is a blank.
	4.11	When hybridisation solution has been added to both slides in a module press the Finished button for the module. Repeat the process for all the additional modules in the Hyb station that require loading, again taking care to note the order in which the modules are made available to avoid mistakes.
	4.12	The program will then proceed automatically and a repeating series of clicks will be heard. These indicate the agitation function is operating correctly, and is opening and closing the valves to facilitate circulation of fluid within the slide chamber.

5.0	POST-HYBRIDISATION TREATMENT OF SLIDES
	5.1	When the hybridisation stations indicate that the reactions are complete, release the clamps and carefully remove the cassette from the module.
	5.2	Gently, lift the cover off and peel the slides from the O-rings. Rack the slides in a staining rack and immerse first in Low Stringency buffer for approximately 1 minute followed by isopropanol for 1 minute.
	5.3	Rack the slides in the slide drying boxes with equal amounts in each box. If there are an odd number of slides, add a polylysine slide to the box with one less slide to ensure the boxes are correctly balanced before placing in the centrifuge. Dry the slides by centrifuging in the Beckman GS-3 centrifuge at 1200rpm for six minutes. The slides should then be racked in a slide box and are ready for scanning.
	5.4	After use a Machine cleaning cycle should ALWAYS be run. Refer to SOP025.00 for the details of the maintenance required at this point. After a machine cleaning cycle has been run on the hybridisation station the O-rings should be removed from the cover of the cassette since it is easier to remove them before the cassette dries out. Soak the O-rings and the cassette cover in a beaker of MilliQ water to remove any wash solution remaining.
	5.5	For Hybridisation of cDNA labels use protocol Hyb2
		O ring conditioning Introduce Probe Hyb Medium Stringency wash High Stringency wash Post wash buffer	75C 60C 42 C 42 C 37 C 37 C	2 minutes 16 hours Flow 20s Flow 20s Flow 20s	Agitate Hold 30s Hold 30s Hold 30s	5 cycles 5 cycles 5 cycles