Hybridizing Cy-labeled cDNA Targets to the TOM1 Microarray

Modified by Rob Alba on 10/10/05

What follows is the protocol I use for pre-hybridization, hybridization, washes, and subsequent scanning of our TOM1 microarrays. The pre-hybridization and wash protocols derive from the lab of John Quackenbush at TIGR ( http://atarrays.tigr.org/PDF/Probehyb.pdf), with a few modifications.

Reagents/Materials Required

1. Pre-Hyb Soln, (5X SSC, 0.1% SDS, 1% BSA)
   1. Filter pre-hyb solution using a 0.2µm SFCA filter unit
2. Wash Soln #1 (1X SSC, 0.2% SDS; pre-heat to 43^oC)
   1. Filter wash solution using a 0.2µm SFCA filter unit
3. Wash Soln #2 (0.1X SSC, 0.2% SDS; room temperature)
   1. Filter wash solution using a 0.2µm SFCA filter unit
4. Wash Soln #3 (0.1X SSC; room temperature)
   1. Filter wash solution using a 0.2µm SFCA filter unit
5. Microarray Hyb Soln (I prep 100 µl per array hybridization)
   1. Livesey Hyb Soln (50µl de-ionized formamide, 25µl 20X SSC, 10µl 50x Denhardt's soln, 5µl 10% SDS, 2.5 µl 0.2M monobasic K-phosphate, 7.5 µl sterile Milli-Q^® H₂0)
   2. NCSU Hyb Soln (33 µl Milli-Q^® H₂O, 30 µl de-ionized formamide, 25 µl 20x SSC, 10 µl 50x Denhardt's, 1 µl 10 µg/ µl PolyA RNA, 1 µl 10% SDS).
6. Isopropyl alcohol
7. 0.1% SDS
8. Milli-Q^® H₂0
9. Coplin staining jars
10. LifterSlips^TM (Erie Scientific Co.; catalog #22X50I-2-4711)
11. 50mL Falcon^TM tubes (place the cap from a 14mL Falcon^TM culture tube at the bottom of each 50mL Falcon^TM tube to elevate the arrays during centrifugation)
12. Corning Hybridization Chambers (catalog #2551)

1. Pre-heat ~60mL of the filtered Pre-Hyb Soln to 43^oC.
2. Incubate arrays in warm Pre-Hyb Soln for 45min.
3. Rinse the "blocked" arrays via 10 dips in Milli-Q^® H₂0, followed by 10 dips in isopropyl alcohol, followed by 10 dips in fresh Milli-Q^® H₂0.
4. Quickly dry the rinsed arrays by centrifugation (30 sec; 1500 rpm) in a 50mL Falcon^TM tube.
   1. Do not let arrays dry out prior to centrifugation.
   2. Excessive centrifugal force will crack array slides.
   3. Use arrays immediately after pre-hybridization.
5. During the pre-hyb step, wash and rinse a sufficient number of LifterSlips^TM.
   1. 5-10 dips in Milli-Q^® H₂O.
   2. 5-10 dips in EtOH.
   3. Dry the clean LifterSlips^TM on lint-free a Kimwipe^®.

1. Conduct a spec assay to asses each Cy-cDNA labeling reaction using the procedure described in TIGR's SOP #M004 (http://pga.tigr.org/sop/M004_1a.pdf). Calculate the total pmol of synthesized cDNA, the total pmol of incorporated Cy dye for each labeling reaction, and the nucleotide/dye ratio for each reaction. Optimal labeling reactions generate >6000 pmols of cDNA (per 60 µl), >200 pmol of Cy dye (per 60 µl), and a nucleotide/dye ratio that is <30.
2. For each Cy-labeled cDNA, calculate the volume of Cy5-cDNA and Cy3-cDNA equivalent to 25-50pmol of incorporated Cy5 or 25-50pmol of incorporated Cy3.
3. Combine the volumes calculated in step 2 in a microfuge tube and minimize the volume using a roto-evaporator (45^oC).
   1. Avoid drying the Cy-cDNA samples completely.
4. Re-suspend the combined cDNA targets in 75 µl of Hyb Soln.
5. Incubate the re-suspended cDNA targets at 95^oC for 5min
6. Centrifuge at max speed for 1 minute (room temperature).
7. Place "blocked" array in a Corning Hybridization Chamber; fill humidity wells as per the manufacturer's instructions.
8. Carefully pipette 63 µl of re-suspended cDNA targets onto array surface; ensure that no bubbles are present.
9. Carefully cover the "loaded" array with a clean dry LifterSlip^TM.
10. Seal the array chamber (without moving the LifterSlip^TM) as per the manufacturer's instructions.
11. Incubate the sealed chamber containing array at 43^oC for 12-16 hours.
    1. Conduct the hybridization in the dark.

1. Fill two foil-covered Coplin jars with pre-heated Wash Soln #1.
2. Remove the LifterSlip^TM from the array surface by dipping arrays in the first Coplin jar containing Wash Soln #1; the LifterSlip^TM should slide off the array easily.
3. Place the array in the second Coplin jar containing pre-heated Wash Soln #1.
4. Incubate arrays in Wash Soln #1 for 10 minutes at 43^oC.
5. Transfer arrays to a new foil-covered Coplin jar containing Wash Soln #2.
6. Incubate arrays in Wash Soln #2 for 10 minutes at room temperature
   1. Agitate arrays gently during wash step.
7. Transfer arrays to a new foil-covered Coplin jar containing Wash Soln #3.
8. Incubate arrays in Wash Soln #3 for 10 minutes at room temperature
   1. Agitate arrays gently during wash step.
9. Immediately dry the washed arrays via gentle centrifugation in a 50mL Falcon^TM tube
   1. Do not let the arrays dry out prior to the centrifugation step.
   2. 30 sec at 1500 rpm
10. Place arrays in foil-covered slide tray until scanning.