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NOTE: The edges of the microarray are etched on the glass slide; always keep track of which side has the DNA.
Breathe on the slide to see the microarray grid appear very briefly.

Use NITRILE gloves when setting up the hybridization reactions; latex gloves contain contaminants that increase nonspecific background on the arrays. Do not touch the surface of the slide, only the edges.

Step 2a: Array Prehybridization

1. Prewarm the microarray (MA) to 50°C for 10 minutes.
2. Prepare Prehybridization Mix:
   
   
   for 25x60mm coverslips
   • 30 µl 2X formamide-based hybridization buffer, which contains bovine serum albumin (BSA) (prewarmed to 70°C for at least 10 minutes or until completely resuspended)
   • 1 µl human Cot-1 DNA (denatured at 95-100°C for 10 minutes prior to use)
   • 29µl nuclease free water
     ---------------------------------------------------
     60 µl Prehybridization Mix
   
   
3. Heat Prehybridization Mix to 80°C for 10 minutes.
   
4. Prepare the coverslip:
   1. put 10 ml 0.2% SDS in a Petri dish and briefly submerge the 25X60mm coverslip with lifters (Erie Scientific).
   2. in a new Petri dish, rinse the coverslip with 10 ml sterile RO water.
   3. blot dry with a KimWipe and use a KimWipe to remove any dust.
   4. place the coverslip, LIFTERS SIDE-DOWN, between the two etches outlining the MA; use a pipet tip to gently align the coverslip.
   
   
5. SLOWLY and CAREFULLY pipet the prehybridization mix at one edge of the coverslip.
   NOTE: capillary action will pull the liquid under the coverslip; you should see it evenly spread until it reaches the other end of the coverslip.
   **The loading must be even and without any bubbles so that the entire array will be exposed to the prehybridization solution.
6. Carefully place the array, DNA side up, inside a 50 ml centrifuge tube containing 100 µl of RO water; put on the tube cap and incubate at 50°C for 1-2 hours (place the tube on its side).
7. Place the MA slide in room temperature 2X SSC/0.2% SDS for 15 seconds.
   The cover slip should just slide off (SAVE the coverslips).
   
   
8. Wash the MA in 2X SSC/0.2% SDS at 60°C for 15 minutes, with gentle rocking.
   
   IMPORTANT: There must be no residual SDS on the MA slide; DO NOT MIX UP THE WASH JARS.
   
   
9. Wash the MA in 2X SSC at room temp. for 10 minutes, with gentle rocking.
10. Wash the MA in 0.2X SSC at room temp. for 10 minutes, with gentle rocking.
    
    Keep the MA slide in the final wash solution until you put it in the ArrayIt Microarray High Speed Centrifuge.
    The slide must be centrifuged immediately after removing from the solution to prevent uneven drying; these "spots" lead to high background.
    
    
11. Gently tap slide on KimWipe to blot off excess wash solution.
    
    Avoid contact with the array surface.
    
12. Insert array side-up into open end of slide holder; make sure slide is fully inserted (slide will drop down into place).
13. Close lid and centrifuge for 10 seconds (2,000xg = 6,000 rpm); press safety latch to stop centrifuge.
14. Open lid and apply slight UP pressure on underside of array and slide out; wipe inside of dome with KimWipe.
15. Proceed to cDNA Hybridization.

Step 2b: cDNA Hybridization

1. Prepare the cDNA Hybridization Mix for EACH array (2 tubes TOTAL) in nuclease free 0.5 ml tubes:
   
   
   for 25x60mm coverslips
   • 12.7µl cDNA #1
   • 12.7µl cDNA #2
   • 2.6 µl nuclease free water
   • 2 µl LNA dT Blocker (3DNA Array 900 Kit)
   • 30µl 2X formamide-based hybridization buffer
     -----------------------------------------------------------
     60 µl cDNA Hybridization Mix
   
   
   
2. Gently vortex and pulse spin the hybridization mix.
3. Incubate hybridization mix at 75-80°C for 10 minutes, and then at 42°C (hybridization temperature) until loading the array.
4. Prewarm the MA and the bottom half of the hybridization chamber to 42°C for 10 minutes.
5. Add 50 µl sterile RO water to each end of the hybridization chamber.
6. Place the MA in the chamber ARRAY SIDE-UP; use a pipet tip to center the slide.
7. Put 10 ml 0.2% SDS in a Petri dish and briefly submerge the 25x60mm coverslip with lifters; in a new Petri dish, rinse the coverslip with 10 ml sterile RO water; blot dry with a KimWipe.
8. Use a KimWipe to remove any dust and place the coverslip, LIFTERS SIDE-DOWN, between the two etches outlining the MA; use a pipet tip to gently align the coverslip.
9. Gently vortex and pulse spin the hybridization mix.
10. SLOWLY and CAREFULLY pipet the hybridization mix at one edge of the coverslip.
    (leave behind any precipitate at the bottom of the tube.)
    NOTE: capillary action will pull the liquid under the coverslip; you should see it evenly spread until it reaches the other end of the coverslip.
    **The loading must be even and without any bubbles so that the entire array will be exposed to the cDNA samples.
    
    
    *If necessary, use a pipet tip to re-center the MA slide inside the chamber.
    **The slide must be centered in order to have proper sealing with the rubber gasket.
    
    
11. Put the cover on the chamber and FINGER-TIGHTEN the screws (OVER-tightening will strip the threads).
12. Cover the hybridization chamber with foil.
    
    KEEP THE HYBRIDIZATION CHAMBER LEVEL AT ALL TIMES!!
    
    
13. Place the chamber in the 42°C hybridization oven OVERNIGHT.
14. Proceed to Post cDNA Hybridization Washes.

****NOTE: You will have to come in by 9 am the NEXT day to perform post cDNA hybridization washes, set up the 3DNA hybridization, and perform post 3DNA hybridization washes.****

Step 2c: Post cDNA Hybridization Washes

1. Carefully remove the foil and the cover from the chamber.
2. With forceps, carefully lift the end of the slide without touching the array.
   
3. Wash the MA in 2X SSC/0.2% SDS (prewarmed to 42 °C) for 10 minutes, with gentle rocking.
   
   The cover slip should just slide off (SAVE the coverslips).
   IMPORTANT: There must be no residual SDS on the MA slide; DO NOT MIX UP THE WASH JARS.
   
4. Wash the MA in 2X SSC at room temp. for 10 minutes, with gentle rocking.
5. Wash the MA in 0.2X SSC at room temp. for 10 minutes, with gentle rocking.
   
   Keep the MA slide in the final wash solution until you put it in the ArrayIt Microarray High Speed Centrifuge.
   The slide must be centrifuged immediately after removing from the solution to prevent uneven drying; these "spots" lead to high background.
   
   
6. Gently tap slide on KimWipe to blot off excess wash solution.
   
   Avoid contact with the array surface.
   
7. Insert array side-up into open end of slide holder; make sure slide is fully inserted (slide will drop down into place).
8. Close lid and centrifuge for 10 seconds (2,000xg = 6,000 rpm); press safety latch to stop centrifuge.
9. Open lid and apply slight UP pressure on underside of array and slide out; wipe inside of dome with KimWipe.
10. Proceed to 3DNA Hybridization.
    

Step 3a: 3DNA Hybridization

1. Prepare the 3DNA Hybridization Mix for EACH array (2 tubes TOTAL) in nuclease free 0.5 ml tubes:
   
   
   for 25x60mm coverslips
   • 2.5 µl Cy3 3DNA Array 900 Capture Reagent
   • 2.5 µl Cy5 3DNA Array 900 Capture Reagent
   • 30µl 2X formamide-based hybridization buffer plus anti-fade reagent
   • 25 µl nuclease free water
     ----------------------------------------------------------------------------------------
     60 µl 3DNA Hybridization Mix
   
   
2. Gently vortex and pulse spin the 3DNA hybridization mix
3. Incubate hybridization mix at 75-80°C for 10 minutes, and then at 50°C (hybridization temperature) until loading the array.
4. Prewarm the MA and the bottom half of the hybridization chamber to 50°C for 10 minutes.
5. Add 50 µl sterile RO water to each end of the hybridization chamber.
6. Place the MA in the chamber ARRAY SIDE-UP; use a pipet tip to center the slide.
7. Put 10 ml 0.2% SDS in a Petri dish and briefly submerge the 24x60mm coverslip with lifters; in a new Petri dish, rinse the coverslip with 10 ml sterile RO water; blot dry with a KimWipe.
8. Use a KimWipe to remove any dust and place the coverslip, LIFTERS SIDE-DOWN, between the two etches outlining the MA; use a pipet tip to gently align the coverslip.
9. Gently vortex and pulse spin the hybridization mix.
10. SLOWLY and CAREFULLY pipet the hybridization mix at one edge of the coverslip.
    (leave behind any precipitate at the bottom of the tube.)
    NOTE: capillary action will pull the liquid under the coverslip; you should see it evenly spread until it reaches the other end of the coverslip.
    **The loading must be even and without any bubbles so that the entire array will be exposed to the 3DNA reagent.
    *If necessary, use a pipet tip to re-center the MA slide inside the chamber.
    **The slide must be centered in order to have proper sealing with the rubber gasket.
    
    
11. Put the cover on the chamber and FINGER-TIGHTEN the screws (OVER-tightening will strip the threads).
12. Cover the hybridization chamber with foil.
    
    KEEP THE HYBRIDIZATION CHAMBER LEVEL AT ALL TIMES!!
    
    
13. Place the chamber in the 50°C hybridization oven for 4 hours.
14. Proceed to Post 3DNA Hybridization Washes.

Step 3b: Post 3DNA Hybridization Washes

• Perform washes in the dark (i.e., cover with foil) to avoid photobleaching and fading of the fluorescent dyes.
• DTT (0.1 mM) in the first two washes reduces fading of Cy5.
• All wash solutions contain deionized, reagent grade water; MilliQ water damages Cy5, and DEPC is a potent oxidizer.

1. Carefully remove the foil and the cover from the chamber.
2. With forceps, carefully lift the end of the slide without touchingthe array.
3. Place the MA slide in room temperature 2X SSC/0.2% SDS for 15 seconds.
   The cover slip should just slide off.
   
   
4. Wash the MA in 2X SSC/0.2% SDS/0.1 mM DTT (prewarmed to 65°C) for 10 minutes, with gentle rocking.
   
   IMPORTANT: There must be no residual SDS on the MA slide; DO NOT MIX UP THE WASH JARS.
   
   
5. Wash the MA in 2X SSC/0.1 mM DTT at room temp. for 10 minutes, with gentle rocking.
6. Wash the MA in 0.2X SSC at room temp. for 10 minutes, with gentle rocking.
   
   Keep the MA slide in the final wash solution until you put it in the ArrayIt Microarray High Speed Centrifuge.
   The slide must be centrifuged immediately after removing from the solution to prevent uneven drying; these "spots" lead to high background.
   
   
7. Gently tap slide on KimWipe to blot off excess wash solution.
   
   Avoid contact with the array surface.
   
8. Insert array side-up into open end of slide holder; make sure slide is fully inserted (slide will drop down into place).
9. Close lid and centrifuge for 10 seconds (2,000xg = 6,000 rpm); press safety latch to stop centrifuge.
10. Open lid and apply slight UP pressure on underside of array and slide out; wipe inside of dome with KimWipe.
11. IMMEDIATELY put the MA slide into a light-protective, low humidity slide box.

Copyright, Acknowledgements, and Intended Use
Created by B. Beason (bbeason@rice.edu), Rice University, 5 June 2002
Updated 26 March 2007