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Malaysia University |
DNA Microarray Hybridization
Contributor:
Chris Seidel
Overview
DNA printed in an array on microscope slides can be visualized by hybridizing the DNA array to labeled fluorescent-Cy cDNA probes prepared by reverse tanscription of mRNA (see Protocol ID#2216). The method described below is a description of how to hybridize Cy cDNA probes to the DNA array.
Procedure
A. General Preparations and Comments for Array Hybridization
Cover slips should be washed thoroughly with 70% Ethanol and then with ddH2O to remove any dust.
Inspect the Hybridization Chamber for cracks around the O-rings and make sure that the chamber is free of dust.
B. Probe Preparation for Array Hybridization
1. On wet rice in the order listed below, add each of the specified items to the Cy-labeled cDNA probe to the following final concentrations (use the cDNA probe prepared in Protocol ID#2216 ; also see Hint #1):
3X SSC (final concentration)
10 μg of PolyA (final concentration)
0.2% SDS (final concentration; also see Hint #2)
2. Denature the cDNA at 95°C for 2 min.
3. Centrifuge briefly in a microcentrifuge at maximum speed to bring any condensation to the bottom of the tube (see Hint #3).
4. The Cy-labeled cDNA Probe mixture is ready to be used for microarray slide hybridization.
C. Microarray Hybridization
1. Place the DNA Array slides in a hybridization chamber.
2. Align the cover slips over the DNA array attached to the slide (see Hint #4).
Regular Cover Slips (see Hint #5)
3a. Pipette between 10 to 12 μl of Cy-labeled cDNA Probe mixture onto the DNA array (prepared in Protocol ID# 2216).
3b. Use a pair of fine forceps to gently place the cover slip on top of the DNA array.
Lifter Cover Slips (see Hint #6)
3a. Place the pipette tip along one of the open edges of the cover slip (see Image #1).
3b. Slowly pipette approximately 20 μl of Cy-labeled cDNA Probe mixture onto the DNA array (prepared in Protocol ID# 2216).
3c. Allow capillary action to draw the Cy cDNA Probe mixture under the cover slip.
D. Chamber Assembly and Array Hybridization
1. Place the slide in the hybridization chamber.
2. Pipette 10 μl of 3X SSC near the edge of the slide away from the cover slip (see Image #2 and Hint #7).
3. Place the cover over the hybridization chamber and tighten the sealing screws (see Hint #8).
4. If more than one hybridization will be performed, properly label each hybridization chamber.
5. Gently place the chamber into a 65°C water bath (see Hint #9).
6. Incubate the hybridization chamber at 65°C for 4 to 12 hr (the contributor of this protocol states that 8 hr is optimal).
E. Chamber Disassembly and Array Washing
1. Prepare Washing Buffers #1, #2, and #3 and ensure that the wash chambers, slide racks, and vacuum centrifuge are ready and available.
2. Add Washing Buffer #1, #2, and #3 to individual washing containers (i.e., Coplin Jars).
3. Carefully remove the hybridization chamber and dry it with paper towels (see Hint #10).
4. Quickly open each of the hybridization chambers and place the array slide into a slide tray suitable for placing in the Coplin jar.
5. Gently dip the slide rack in and out of Washing Solution #1 (see Hint #11).
6. Gently dip the slide rack in and out of Washing Solution #2 several times.
7. Gently dip the slide rack in and out of Washing Solution #3 several times (see Hint #12).
8. After the last wash, place the slide rack into the vacuum centrifuge and centrifuge with vacuum for 5 minutes at 500 rpm (see Hint #13).
9. Remove the slides and place them into a dust-free box for scanning.
Solutions
10% (w/v) SDS
PolyA
1 mg/ml poly-dATP
SSC (20X)
6 M NaCl
pH 7.2
0.6 M Sodium Citrate
Wash Solution #1
Wash Solution #2
297 ml of ddH2O
3 ml of 20X SSC
Wash Solution #1
900 μl of 10% SDS
750 μl of 20X SSC
284 ml of ddH2O
15 ml of 20X SSC
300 ml of ddH2O
BioReagents and Chemicals
SDS
Sodium Citrate
Sodium Chloride
poly-dATP
Protocol Hints
1. The Cy-labeled cDNA Probe can be dried in a vacuum centrifuge to an appropriate volume for adding probe mix components.
2.To avoid precipitation of the SDS, add it last just prior to denaturation. Also, do not be overly concerned with accurately pipetting very small volumes (μl) of SDS.
3. It is important to rapidly cool the probe to room temperature before pipetting it onto the array; however, do not place it on ice because the SDS will precipitate. Some microcentrifuges allow the tube to spin through air (i.e., the back end of the microcentrifuge tube is not protected by a cover). These microcentrifuge types are ideal since the moving air rapidly cools the tube and its contents to room temperature.
4. Confirm that the slide is right side up relative to the DNA array attached to the slide. The DNA spots can usually be visualized by breathing on the slide to fog the surface. Little spots of DNA can be seen as the fog evaporates.
5. Regular cover slips are the standard run-of-the-mill style cover slips that have been around for years. The process of adding solution to the slide and placing the cover slip on the slide without introducing air bubbles is worth practicing when using regular cover slips.
6. Lifter cover slips have a little edge of Teflon along two opposite edges of the cover slips. These lifter slips leave a very small space between the slide and the cover slip that can be filled, as in this case, with a solution. The introduction of air bubbles between the cover slip and the slide is not a problem since the air bubbles often disappear during hybridization. Nevertheless, try not to introduce air bubbles.
7. The extra 3X SSC keeps the chamber humidified during incubation.
8. Be careful not to bump or jar the chamber. Do not allow the hydration drop to merge with the cover slip and dilute the probe. Likewise, do not allow the cover slip and probe to bump into the sides of the chamber and wick out along any surfaces.
9. Be careful not to burn yourself. Acting swiftly but gently will help prevent major problems. Putting on two gloves can help insulate your hands from the water. Alternatively, use a tool or rack for placing the chambers under water, or another heat source, such as a hot bonnet PCR machine or incubation oven may be used.
10. If there are several hybridization chambers to process, dry each and remove the screws. Do not open the covers until all chambers are dried and ready. The time that elapses from the moment the chamber is opened to the time the slide rack is immersed in the first wash should be minimized to prevent drying out of the probe. The slide chambers contain a small recess under one edge of the slide for prying the slide out of the chamber. Open the slide chamber and place the slide in a slide rack.
11. Allow the cover slip to fall off without scratching the surface of the slide or getting stuck on the slide rack. Some slide racks are constructed in such a way that it is very difficult to get the cover slip to fall free of the slide. Wheaton Slide racks are recommended because they contain no obstruction in the vicinity of the cover slip. Be careful not to let the surface of the slide dry at any time during the washes. Once the cover slip is free, a few plunges up and down are sufficient to wash the slide.
12. The aggregate time for all three washes is usually less than five minutes.
13. Allow the surface of each slide to evenly dry in order to avoid background problems. It is extremely important that a centrifuge capable of handling a microscope slide rack is used and is immediately available after the last wash.
