Preparation of Fluorescent DNA Probe from mRNA: Human

Probe Labeling and Hybridization

Preparation of Fluorescent DNA Probe from mRNA: 32 Sector Human Arrays (Hale protocol 1/9/00)

To anneal primer, mix 2ug of mRNA with 4ug of random nanomer in a total volume of approximately 15ul. The volumes used are variable depending on the concentration of the mRNA sample. Do not attempt to concentrate the mRNA sample by microcentricon as too much of it will be retained by the filter. Do not concentrate the mRNA by drying it down if it is in a buffer that contains salts like Fast Track's Elution Buffer.

	Cy3	Cy5	Blank
mRNA (1 ug/ul)	2 ul	2 ul	blank
Random Nanomer (1 ug/ul)	4 ul	4 ul	Concentration is variable
ddH₂O (DEPC) or Fast Track Elution Buffer	~7 ul	~7 ul	Actual volume depends on concentration of mRNA sample
Total volume:	15 ul	15 ul

Heat to 65C for 10 min and cool on ice.
Add 15 uL of reaction mixture each to Cy3 and Cy5 reactions:

Reaction mixture	ul	Unlabeled dNTPs	Vol.	Final conc.
5X first-strand buffer*	6.0	dATP (100 mM)	25 uL	25 mM
0.1M DTT	3.0	dCTP (100 mM)	25 uL	25 mM
Unlabeled dNTPs	0.6	dGTP (100 mM)	25 uL	25 mM
Cy3 or Cy5 (1 mM, Amersham)	3.0	dTTP (100 mM)	10 uL	10 mM
Superscript II (200 U/uL, Gibco BRL)	2.0	ddH2O	15 uL
ddH₂O (DEPC) or Fast Track Elution Buffer	5.4		-----
Total volume:	25 ul	Total volume:	100 uL

* 5X first-strand buffer: 250 mM Tris-HCL (pH 8.3), 375mM KCl, 15mM MgCl2)

Incubate at 42C for 1 hr.
Add 1 ul SSII (RT booster) to each sample. Incubate for an additional 0.5-1 hrs.
Degrade RNA by addition 15ml of 0.1N NaOH, and incubate at 70^oC for 10 min.
Neutralize by addition of 15ml of 0.1N HCl, and bring the volume to 400ml with TE (10mM Tris, 1mM EDTA). Combine both probes into 1 centricon. [WASH 1] Spin 11 min. at 12kRPM in a Centricon-30 micro-concentrator (Amicon). If repurification of cy dye flow-through is desired, do not combine probes until Wash 2.
WASH 2: Add 450 ul 1X TE and spin 11 min. at 12kRPM.
WASH 3: Add 450 ul 1X TE, 20 mg of Cot1 human DNA (Gibco-BRL), 2mL of 10m g/ml polyA RNA (Sigma, #P9403) and 2 ml of 10mg/ul tRNA (Gibco-BRL, #15401-011). Spin 15 min. at 12 kRPM. Look for nice concentration of the "colored probe" in the centricon. Try to concentrate to a volume of less than 10 ml. This volume is attained when the center of the membrane is dry and the probe forms a ring of liquid at the edges of the membrane.
Invert the centricon into a clean tube and spin briefly at 14kRPM to recover the probe. 10 minutes is enough but less time may be adequate for good recovery.

For 22 mm x 40 mm arrays:….

Adjust volume to 20ml with 1X TE. (12 ml for 22 mm x 22 mm arrays)

For final probe preparation add 4.25l 20XSSC (3.0 M NaCl, 300 mM NaCitrate (pH8.0)) and 0.75l 10%SDS (2.55 ml 20X SSC & 0.45 ml 10% SDS for 22 mm x 22 mm arrays). When adding the SDS, be sure to wipe the pipette tip with clean, gloved fingers to rid of excess SDS.

Denature probe by heating for 2 min at 100^oC, and incubate at RT for 15-20 min.

Spin at 14kRPM for 10 min.

Place 24 ml probe on the array under a 22mm x 40mm glass cover slip (12 ul for a 22 mm x 22 mm cover slip).

To maintain the proper humidity, spot about 5

ml 3X SSC at each corner of the slide, as far away from the array as possible. If using a Gene Machines hyb chamber, or similar apparatus, put drops of 3X SSC under the slide in the recessed troughs.

Hybridize in a heated water bath at 65 C for 14 to 18 hours. The hyb chamber should not be in direct contact with the walls of the water bath or else it may overheat. Ideally, the same hybridization conditions, including time in the water bath, should be used for all chips in a study.

Ready washes in 250 ml chambers to 200 ml volume [2X SSC (Wash I) with 0.1%SDS (add this to the wash first), followed by 1X SSC (Wash II -- prepare two of these chambers), and 0.2X SSC (Wash III)]. Avoid adding excess SDS. The Wash I chamber and one of the Wash II chambers should each have a slide rack ready.

Blot dry chamber exterior with towels and aspirate any remaining liquid from the water bath. Be sure to aspirate in between the two chamber halves. Aspiration may not be necessary or possible if using the Gene Machines hyb chamber.

Unscrew chamber; aspirate the holes to remove last traces of water bath liquid. This is not an issue with the Gene Machines hyb chamber as the thumb screws cannot be removed from the cover.

Place arrays, singly, in rack, inside Wash I chamber (maximum 4 arrays at a time). Allow cover slip to fall off by itself. Manually removing the coverslip with forceps or a razor blade is risky, but can be executed successfully. DO NOT AGITATE until cover slip is safely removed. Then agitate for ~15 sec by plunging the rack up and down in the chamber. A good foam should be developed and run over the sides of the wash chamber - this is supposed to be messy! Failure to vigorously wash the rack in this step will allow excessive amounts of unbound probe to stick to the array. During this procedure it may be necessary to hold the slides in the rack with the clean, gloved fingers of one hand while the other plunges the rack.

Remove the chips by grasping the slide on one end (far away from the array) with clean, gloved fingers. Rinse the slides briefly one by one in a Wash II chamber without a rack, and transfer to the Wash II chamber containing a new rack. This step minimizes transfer of SDS from Wash I to Wash II. This is important for minimizing background. Again, this should leave wash solution on your bench.

Wash arrays by submersion and agitation for 30 sec. in Wash II chamber, then Wash III (transfer the entire slide rack this time). Your bench should now be a sopping mess.

Carry your rack of slides in the Wash III chamber to a suitable centrifuge and "Spin dry" by centrifugation of the rack in a Beckman GS-6 tabletop or comparable centrifuge at 600 RPM for 4 min. at room temperature. Make sure you use a second rack with an identical number of blank microscope slides to balance the rack with your arrays.

Scan arrays immediately. A delay of up to an hour or so will not ruin your slides, but note that the dye is reasonably unstable in this state and the measured ratios can change significantly within hours.