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RNA labelling

1. Prepare total RNA at a concentration of 2ug/ul or more in DEPC-treated water. Mix 25ug total RNA, 1.25ul dNTP mix (25mM dATP, dTTP, and dGTP; 10mM dCTP), 1ul anchored oligo-dT (100uM; SigmaGenosys), and DEPC-treated water to a total of 12.5ul.
2. Heat at 65 degrees C for five minutes.
3. Add 5ul 5x reaction buffer (supplied with reverse transciptase), 1.25ul DTT (ditto, 0.1M), 1ul reverse transciptase (Superscript III, Invitrogen), and 3ul Cy5 or Cy3 (Amersham). ****Keep tubes in darkness from this point on as much as possible*** Mix well and spin down any condensation inside the tube.
4. Heat at 50 degrees C for two hours.
5. Add 1.5ul 1M NaOH. Heat at 70 degrees C for five minutes.
6. Add 1.5ul 1M HCl.
7. Prepare a Autoseq G-50 column (Amersham); mix contents by quickly vortexing, snap off the bottom of the assembly, unscrew the lid 1/4 of a turn, place in a 2ml eppendorf tube and spin for 1 minute at 200rpm. Discard the 2ml tube and place the column in a 1.5ml tube.
8. Pipette the labelled sample onto the centre of the G-50 resin (be careful to avoid the liquid from passing down the inside of the column without entering the resin). Spin for 1 minute at 2000rpm, then discard the column.
9. Add the flow-through to the corresponding one with which it is to be compared (labelled with the complementary dye).
10. Add one-tenth volume of 3M NaOAc pH 5.2 and 3 volumes 100% EtOH. Precipitate at -70 degrees C for 30 minutes.

Hybridisation

Optionally at this stage one can prehybridise slides for 1-2 hours, using the hybridisation mix below, and afterwards briefly washing them before addition of sample. We find that this can help to reduce background.

1. Spin the ethanol precipitated labelling reaction at 13000rpm for 30 minutes (noting which side of the tube is towards the outside of the rotor), then pipette away the supernatant. Add 100ul of 70% EtOH, then spin for 10 minutes with the tube in the same orientation within the rotor.
2. Air dry for 5 minutes at room temperature. Add 37ul hybridisation mix (30ul hybridisation buffer [5x SSC, 6x Denhardt's, 60mM TrisHCl pH 7.6, 48% formamide; filter sterilised] plus 3.5ul polyA DNA [Sigma; 2ug/ul] and 3.5ul yeast tRNA [Sigma]), and resuspend pellet using pipette.
3. Heat at 100 degrees C for five minutes, then centrifuge at room temperature at 13000rpm for three minutes.
4. Clean a 25x60mm coverslip of dust with an airgun, and pipette the sample in a line along the centre of the length of the coverslip, avoiding making bubbles. Similarly clean the microarray slide to be used, holding it by its label. Place the slide label-side down (hence also printed side down) onto the coverslip so that one end of the coverslip extends beyond the top end of the slide (the end further away from the label).
5. Place a piece of 3M paper into a Genetix hybridisation chamber and dampen it with 1.5ml 15x SSC.
6. Insert the slide into the chamber coverslip up, ensuring that the coverslip is pushed fully over the slide, with its edge level or close to being level with the top edge of the slide.
7. Place in an incubator overnight at 42 degrees C (15-18 hours).

Post-hybridisation washing

Washing can be done in either histology wash boxes or alternatively 50 ml Falcon tubes (We have found we obtain cleaner results with Falcon tubes, placing one slide/tube and using a fresh tube for each wash solution)

1. Carefully remove slide from hybridisation chamber and place in a coplin jar containing wash solution 1 (2x SSC, 0.4% SDS,room temperature; all washes take place at room temperature) gently agitate until coverslip slides off.
2. Place slide in wash solution 1 for 10 minutes
3. Place slide in fresh wash solution 1 for 6 minutes
4. Place slide in wash solution 2 (0.4 x SSC) for 2 minutes
5. Repeat wash in fresh solution 2 for 1 minute
6. Place slide in wash solution 3 (0.1 x SSC) for 1 minute
7. Repeat wash in fresh solution 3 for 30 seconds
8. "Dip" slides in solution 4 (0.01 X SSC) for 30 seconds
9. Centrifuge slides ~1200 rpm for 4 minutes
10. Scan slides........
    

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