Hybridizing Cy-labeled cDNA Targets to the TOM2 Oligo-array

Modified by Ryan McQuinn

What follows is the protocol I use for pre-hybridization, hybridization, washes, and subsequent scanning of our TOM2 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. Hybridization buffer from NC state.

Reagents/Materials Required-(*made fresh each time)

  1. *Pre-Hyb Soln, (5X SSC, 0.1% SDS, 1% BSA)
  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 (calculated per rxn)
    1. NCSU Hyb Soln : per rxn-formamide-13.5µl, 20x SSC 11.25µl, 50x denhardt's 4.5µl, PolyA (10µg/µl) 0.45µl, water 14.85µl, 10% SDS 0.45µl)
  6. Isopropyl alcohol
  7. 0.1% SDS
  8. Milli-Q® H2O
  9. Coplin staining jars
  10. LifterSlipsTM (Erie Scientific Co.; catalog #22X50I-2-4711)
  11. 50mL FalconTM tubes (place the cap from a 14mL FalconTM culture tube at the bottom of each 50mL FalconTM tube to elevate the arrays during centrifugation)
  12. Corning Hybridization Chambers (catalog #2551)
  13. 0.1% SDS
  14. 100% Ethanol
Treatment of slides prior to prehybridization (immediately prior to prehybridization):
  1. Rehydrate slide (barcode side up) over a 65 oC waterbath
  2. UV treat slides at 65 mJ
  3. Move on immediately to Pre-hybridization step
Pre-hybridization/Blocking the Arrays
  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® H2O, followed by 10 dips in fresh Milli-Q® H2O, followed by 10 dips in isopropyl alcohol.
  4. Quickly dry the rinsed arrays by centrifugation (30 sec; 1500 x rpm) in a 50mL FalconTM 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 LifterSlipsTM.
    1. 5-10 dips in Milli-Q® H2O.
    2. 5-10 dips in EtOH.
    3. Dry the clean LifterSlipsTM on a lint-free Kimwipe
Preparation of Cy-Labeled cDNA Targets
  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 60ul), >200 pmol of Cy dye (per 60 ul), 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 200 pmol of incorporated Cy5 or 200 pmol 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 65µl of Hyb Soln.
  5. Incubate the re-suspended cDNA targets at 95 oC for 4 min
  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 all of re-suspended cDNA targets onto array surface; ensure that no bubbles are present.
  9. Carefully cover the "loaded" array with a clean dry LifterSlipsTM.
  10. Seal the array chamber (without moving the LifterSlipsTM) as per the manufacture's instructions.
  11. Incubate the sealed chamber containing array at 42 oC for hours.
    1. Conduct the hybridization in the dark.
Washing Arrays after Hybridization (better if more wash buffer per array-use medium glass chambers for up to 4 slides)
  1. Fill a glass washing chamber with pre-heated Wash Soln #1.
  2. Remove the LifterSlipsTM from the array surface by dipping arrays in the Plastic Coplin staining jar containing Wash Soln #1; the LifterSlipsTM should slide off the array easily.
  3. (twice) Place the slides in the glass chamber with pre-heated Wash Soln#1 (~43oC)
  4. Incubate arrays in Wash Soln #1 for 3 minutes at RT oC on a Rocker mixer
  5. Transfer arrays using the glass slide holder to a new glass chamber containing Wash soln #2.
  6. Incubate arrays in Wash Soln #2 for 3 minutes at room temperature
    1. Rock arrays gently during wash step again on the Rocker.
  7. Dip slides individually in Wash Soln #3 in a plastic Coplin jar. Then transfer arrays to a new glass chamber containing Wash Soln #3 using the glass slide holder.
  8. Incubate arrays in Wash Soln #3 for 3 minutes at room temperature
    1. Rock arrays gently during wash step.
  9. Repeat step 7 twice in new wash #3 each time (for a total of 3 times).
  10. Dip in Wash Soln #3. Then immediately dry the washed arrays via gentle centrifugation in a 50mL FalconTM tube.
    1. Do not let the arrays dry out prior to the centrifugation step.
    2. 2 min at 1500 x rpm
  11. Place arrays in foil-covered slide tray until scanning.
Scanning Arrays
We scan our arrays immediately after they are washed/dried using a two-channel confocal microarray scanner (ScanArray5000; GSI Lumonics, MA) and the associated ScanArray software (v3.1 Packard Biochip Technologies, MA). After laser focusing and balancing of the two channels, scans are conducted at a resolution of 10µm with the laser power set at 90% of maximum and the photomultiplier tube typically set at 60-85% of maximum.

Excitation/emission settings are 543µm/570µm and 633µm/670µm for the Cy3 and Cy5 fluors, respectively. Raw fluorescence image data is saved as .tif files, which are converted to numerical signal data (.txt files) using ImaGene software (v4.2, BioDiscovery Inc., CA).