Preparation of Fluorescent DNA Probe from mRNA: Human

Preparation of Fluorescent DNA Probe from mRNA: Human (Ash/Max protocol; 8/3/00)

To anneal primer, mix 2ug of mRNA with 4ug of a regular or anchored oligo-dT primer in a total volume of 10ul:

	Cy3	Cy5
mRNA (1 g/l)	2 l	2 l
Oligo-dT (4 g/l)	1 l	1 l	(Anchored: 5'-TTT TTT TTT TTT TTT TTT TTV N-3')
ddH₂O (DEPC)	7 l	7 l
Total volume:	10 l	10 l

Heat to 65^oC for 10 min and cool on ice.

Add 10 uL of reaction mixture each to Cy3 and Cy5 reactions:

Reaction mixture	l	. . . . .	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)	5.4			-----
Total volume:	20 l		Total volume:	100 uL

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

Incubate at 42^oC for 1 hr.
Add 1 l 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 7 min. at 14kRPM 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 7 min. at 14kRPM.

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 7 min. at 14 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.

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

Hybridize at 65^oC for 14 to 18 hours in a custom slide chamber with humidity maintained by a small reservoir of 3X SSC (spot around 3-6 l 3X SSC at each corner of the slide, as far away from the array as possible).

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.

Unscrew chamber; aspirate the holes to remove last traces of water bath liquid.

Place arrays, singly, in rack, inside Wash I chamber (maximum 4 arrays at a time). Allow cover slip to fall, or carefully use forceps to aid cover slip removal if it remains stuck to the array. DO NOT AGITATE until cover slip is safely removed. Then agitate for ~15 sec.

Remove array by forceps, rinse in a Wash II chamber without a rack, and transfer to the Wash II chamber with the rack. This step minimizes transfer of SDS from Wash I to Wash II.

Wash arrays by submersion and agitation for 30 sec. in Wash II chamber, then Wash III (transfer the entire slide rack this time).

"Spin dry" by centrifugation in a slide rack in a Beckman GS-6 tabletop centrifuge at 600 RPM for 2 min

Scan arrays immediately.