1. Prepare a Master Mix (RT)
6 ul 5X First Strand Buffer
3 ul 0.1M DTT
3 ul 10X dNTP Mix (25 mM dGTP, dATP, dCTP, 15 mM dTTP, 10 mM aminoallyl-dUTP)
2 ul Deionized water
2 ul SuperScript Reverse Transcriptase (200 units/ul)
16ul Total Volum
2. 20ug (x ul) RNA
11-x ul ddH2O
2.5 ul Oligo dT
0.5 ul Random Primer Mix
14ul Total Volum-----votex, spin down for 5 seconds
3. 70oC 10min
Chill on ice 1min
Add 16ul Master Mix
Add 0.5ul SuperScript Reverse Transcriptase (200 units/ul)
Add 10 ul 1N NaOH
70oC 10min, spin tubes briefly
Add 25ul 1M Hepes Buffer pH 7.5, then 1ml binding buffer, mix well by pipetting, apply to Zymo columns, spin at 6000 rpm for 30 sec, add 600ul washing buffer and spin at 14,000 rpm for 1min and transfer to a fresh new tube, add 8ul ddH2O, sit down for 1 min and spin for 30 sec, then add 6ul H2O, sit for 1min and spin for 30 sec.
4. DARK PLACE
Add 12 ul Clontech DMSO to the dye vial (Cy3 or Cy5), votex, briefly spin down.
Add 1.5 ul 1M NaHCO3 and 2 ul dye to the cDNA sample, mix well, wrapped by the aluminum foil and store at RT for 60 min.
5. Add 2 ul 3M Sodium Acetate and 50 ul 100% EtOH, votex.Place at 20oC for 2hrs.
6. Centrifuge at maximum speed at 4oC for 20 min. Pipet off supernatant and wash pellet once in 70% EtOH 200ul. Spin for 5min at 4oC. Air dry for 10 min. Dissolve in 100 ul ddH2O.
7. Add 500 ul buffer PB to the sample, pipet well.
8. Prepare the QIAquick spin Columns and collection tubes. Load the sample to the column and spin at maximum speed for 1 min at RT. Discard the flowthrough.
9. Reinsert the column in the collection tubes and add 750 ul Buffer PE to the column and spin at maximum speed for 1 min at RT. Discard the flowthrough. Repeat 1 more time.
10. Put back the column to the collection tubes and spin at maximum speed for 1 min.
11. Place the column in a clean 1.5 ml tubes. Add 30 ul Buffer EB and let stand for 1 min. Spin for 1 min. Add 30 ul Buffer EB and let stand for 1 min. Spin for 1 min. Combine two cDNA sample together. Concentrate in SpeedVac for 1hr-2hr to reduce the amount to 10 ul. Store at 20C wrapped very well.
12. Make 100 ul 2x Hybridization Buffer (20x SSC 40ul, 20% SDS 1ul, PolyA 10ul and ddH2O 49ul), filter with 0.45um syringe filter. Take 10ul for each hybridization probe. Add the labeled probe to the hybridization buffer, mix by pipetting. Heat the probe at 94C for 2 min, then spin for 1 min to cool down.
13. Place the slide into the hybridization chamber, and add 25 ul ddH2O in each well under the slide. Put the lifterSlip on the slide and add the hybridization solution containing the probe between the lifterSlip and the slide. Add 10ul 4x SSC to one end of the glass slide to keep the chamber humidified
14. Close the chamber and submerge in a 62oC water bath.
15. Disassemble the hybridization chambers quickly and place the slides in the RT 2x SSC/0.1% SDS and allow lifterSlip to fall off.
16. Label four Coplin Jars with Wash1a, 1b, 2, 3
Wash 1a: 2x SSC/0.1% SDS (prewarm at 62oC) for 5 min at RT
Wash 1b: 2x SSC/0.1% SDS (prewarm at 62oC) for 5 min at RT
Wash 2: 0.2x SSC at RT for 1 min
Wash 3: 0.05x SSC at RT for 1 min
17. Rinse the slides with distilled water, then use the nitrogen gas to remove the water on the surface or spin the slides at 1,200 rpm for 4min using slides holder. Put the slides in the slides container and ready for scanning.
Scanning with the Axon 4000B scanner