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Technical support : Hybridisation
1. Background information
The protocol described here has been optimised for the hybridisation of a labeled cDNA target (resulting from the mix of Alexa fluor 555- and Alexa luor 647-labeled samples from human origin) onto glass slide spotted with 60-mer oligonucleotides. Using an other type of microarray probe (PCR amplified cDNA, shorter or longer oligonucleotides) will require to empirically adapt the experimental conditions with the aim at increasing the positive signal and decreasing the background fluorescence. Several parameters are critical : buffer stringency, hybridisation temperature, incubation time, adding of adjuvant molecules (poly(A)-DNA, Cot1-DNA, tRNA, BSA...), pre-processing of the slides, post-hybridisation washing.
2. Material
20 x saline-sodium citrate (SSC) : 0.3 M sodium citrate, 3 M NaCl, pH 7.0
20 % sodium dodecyl sulfate (SDS)
Formamide, deionised (Sigma, cat# F-9037)
Human COT1-DNA (Invitrogen, cat# 15279-011)
Salmon sperm DNA (Invitrogen, cat# 15632-011)
Poly(A)-DNA (Amersham, cat# 27-7836-01)
Lifterslip cover glass, M series (Erie scientific, cat# 221x25-2-4635)
Non-powdered gloves
3. Procedure
3.1. Preparation of the hybridisation mixture
Mix equivalent amounts of the two labeled cDNA samples (as determined by spectrophotometry; 0,65-1,3 µg of each cDNA is required). Add 10 µg of Human Cot-1 DNA and 10 µg salmon sperm DNA (optional : 10 µg of poly(A)-DNA).
Add 20 µg of glycogen (2 µL)
Add 12 µL 3 M sodium acetate pH 5.2
Add 350 µL 100 % ethanol
Mix well by inverting the tube.
Incubate for at least 1 hour at -80°C, and then centrifuge at 16.000xg for 20 minutes at 4°C.
Wash the pellet with 750 µL of cold 75 % ethanol, and centrifuge at 16.000xg for 5 minutes at 4°C. Remove the supernatant by careful pipetting (DO NOT invert the tube).
Dry the pellet in a Speed-Vac at 30°C for 5-10 min or until all liquid has been evaporated.
3.2. Hybridisation
Incubate the hybridisation buffer (25 % deionised formamide, 5X SSC, 0.1% SDS) for at least 60 min at 42°C. Vortex several times and centrifuge briefly to remove any particle from the buffer.
Resuspend the Alexa dye-labeled pellet in 25 µL of preheated hybridisation buffer. Mix by pipetting until complete dissolution of the pellet, briefly vortex and centrifuge.
To denature, heat the mixture at 95°C for 3 minutes, and snap cool on ice for 30 sec. Centrifuge the mixture at maximum speed for 1 minute. Use immediately.
Place the (prehybridised) microarray slide in the hybridisation chamber. Using forceps, place a precleaned glass coverslip over the array print area.
Pipette the labeled mixture to the slide surface using a thin pipette tip. Slowly inject the target under one corner of the coverslip until the array surface is covered. Continue to apply remaining target at the other corners.
Add 10 µL of water to the small wells at each end of the chamber, cover, and tightly close the chamber lid.
Incubate the array at 42°C for 18-20 hours using appropriate surface acoustic wave agitation frequency.
Note 1 : Do not touch the spotted area of the microarray with hand at any time. Do not allow the array to dry before the end of the hybridisation.
Note 2 : the hybridisation volume is dependent on the type of glass coverslip that will be used to create the hybridisation micro-chamber on the array (Lifterslip 22x25 mm : 24 µL, Lifterslip 25x60 mm : 64 µL).
Note 3 : Expose the labeled sample to light as little as possible during the hybridisation process.
Note 4 : To prevent bubbles, make sure there is no debris on the array before laying down coverslip. Bubbles may also be caused by not allowing the solution to completely cover the array before injection at a different corner begins.
3.3. Array washing
Prepare wash solutions in glass slide dishes, with each dish having its own rack.
Wash solution 1 : 2x SSC, 0.1% SDS Wash solution 2 : 1x SSC Wash solution 3 : 0.5x SSC
Wash solution 1 is prewarmed at 42°C while solutions 2 and 3 are used at room temperature. All buffers should be filtered using a 0.45 µm membrane.
Carefully remove the array from the hybridisation station, making sure to keep horizontal the chamber level.
Place the hybridised array into a clean slide dishes filled with 200 mL of pre-warmed washing buffer 1. Once submerged, tilt the array and gently dump off the coverslip. It may be necessary to lightly swish the array under solution to dislodge the slip. Continue washing for 10 minutes at room temperature with gentle agitation on an orbital shaker or equivalent.
Wash the microarray in washing buffer 2 for 10 minutes at room temperature as described previously.
Wash similarly the microarray in washing buffer 3 for 10 minutes at room temperature.
Dry the slide by centrifugation at room temperature for 5 minutes at 200 g.
Place the array in a light tight slide box until it can be scanned. Use a plastic box to avoid dust deposition on the slide surface.
Note 1 : do not allow the slide to dry at any time before the end of the procedure. This will cause uneven fluorescent background. Dry the slide by centrifugation at room temperature for 2 minutes at 200 g using either a 50 mL conical-bottom tube or a rotor for microtiter plates.
Note 2 : Streaking or mottling on the slide surface indicates further washing is necessary. Repeat the wash cycle as necessary to clean the slide. Dry the slide between each cycle.
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