Preparation Prior to Starting Protocol

RT Reaction

Preparing the Label

Prehybridization of Microarray

Hybridization

Post-hybridization Washing

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Materials Needed:

• Isolated and quantitated total RNA samples
• Microarray slides (i.e. DNA chips)
• Contents of Genisphere 3DNA Submicro EX kit (“Vials” below refer to kit)
• RNase-free water
• Coverslips, 22 x 40mm size from Corning
• Superscript RT kit from Life Technologies, Inc. (optional)

Small volumes of the following (<1 ml):

• 0.5 M NaOH / 50 mM EDTA
• 1 M Tris-HCl, pH 7.5
• 10 mM Tris-HCl, pH 8.0 / 1 mM EDTA
• 5 mg/ml linear acrylamide (Ambion)
• 3 M Ammonium Acetate
• 100% Ethanol, 70% Ethanol
• 100 ml each of following:
• 2X SSC / 0.1% SDS / 0.1% BSA
• 2X SSC/0.2% SDS
• 2X SSC
• 0.2 X SSC
• 0.2 % SDS
• Liquid nitrogen

Get Ready Beforehand:

• Dry incubator set to hybridization temp (example 50° C)
• Preheat 50 ml of each of the following in dish or Coplin jar to hybridization temp :
  • 2X SSC / 0.1% SDS / 1% BSA
  • 2X SSC/0.2% SDS
  • 50 ml of 2X SSC
  • 50 ml of 0.2X SSC
• Water baths or thermal cycler set to 42° C, 50° C, 65° C, 80° C

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Reverse Transcription (~30 minutes set-up and 2 hour incubation)

1. Set up RNA-RT primer mix for each RNA sample in labeled RNase-free tubes:

• 10 µl of 0.2 µg/µl RNA (total yeast RNA isolate)
• 1 µl of Cy3 or Cy5 RT primer, 0.2 pmol (use a 25 fold dilution of Vial 2)

2. Mix and spin to bottom of tube

3. Heat to 80° C for 10 minutes and place on ice.

4. Set up reaction mix for each reaction (may want to make a master mix and aliquot):

• 4 µl 5X RT buffer (Vial 5)
• 1 µl dNTP mix (Vial 4)
• 3 µl RNase free water
• 1 ul RNasin (40 units)
• 1 µl RT enzyme, 200 units (Vial 3)

Or

5. Incubate 42° C for 2 hours.

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Preparation of Label (~1.5 hours)

1. Stop RT reactions with 3.5 µl of 0.5 M NaOH / 50 mM EDTA. Put reactions in -20° C.

2. Incubate at 65° C for 10 minutes

3. Neutralized with 5 µl of 1 M Tris-HCl, pH 7.5

4. Combine the Cy3 and Cy5 reactions for a given experiment:

• Add the 28.5 µl of Cy3 reaction tube to the Cy5 reaction tube
• Add 43 µl of 10 mM Tris-HCl, pH 8.0 / 1 mM EDTA to Cy3 tube to rinse
• Transfer the 43 µl rinse to the Cy5 tube

• Prepare SCL size exclusion column and load 100 ul from above
• Elute and measure volume (if different from 100 ul, modify next 3 steps)
• Add 2 ul of 5 mg/ml linear acrylamide

5. To the combined Cy3 / Cy5, add 2 µl of 5 mg/ml linear acrylamide

6. Add 250 µl 3 M Ammonium Acetate

7. Add 875 µl 100% Ethanol, mix well

8. Place at –20° C for 30 minutes.

9. Microfuge at >10,000g for 15 minutes

10. Aspirate supernatant

11. Add 300 µl 70% Ethanol to pellet and mix

12. Microfuge at >10,000g for 5 minutes

13. Dry pellet for 30 minutes at 55-65° C

14. Thaw and resuspend Alternate Hybridization Buffer (Vial 7): heat to 50° C, mix well

15. Resuspend pellet in following to prepare probe:

• 23 µl Alternate Hybridization Buffer (Vial 7)
• 2.5 µl Cy3 Capture Reagent (Vial 1)
• 2.5 µl Cy5 Capture Reagent (Vial 1)
• 2 µl of 0.1 ug/µl yeast tRNA
• 1 µl Anti-Fade Reagent (Vial 8)

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Pre-Hybridization (~45 minutes)

1. Place microarray slide in Coplin jar with 50 ml of 2X SSC / 0.1% SDS / 0.1% BSA, preheated to hybridization temp (example 50° C), incubate for 30 minutes

2. Transfer slide to dish or Coplin jar with 50 ml of 2X SSC for 5 minutes at 50° C

3. Transfer slide to dish or Coplin jar with 50 ml of 0.2X SSC for 5 minutes at 50° C

4. Dry slide by placing in 50 ml tube and spinning 2-3 minutes at 500 rpm in clinical centrifuge

5. Prepare coverslip by dipping into 0.2 % SDS, then water. Blot and let dry. Proceed to hybridization steps below.

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Hybridization (3 days)

1. Add the 31 µl of probe to the microarray slide on one end between the etched corners

2. Place coverslip on same end and let fall slowly. Probe should spread out with no bubbles

3. Transfer slide to 50 ml tube

4. Keep the slide and tube horizontal and add 300 µl water

5. Seal tube with cap and parafilm

6. Place in dry incubator at hybridization temp (example 50° C) for 3 days

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Post-Hybridization (~1 hour)

1. Place microarray slide in a Coplin jar at room temp with 2X SSC/0.2% SDS for 15 seconds. When removing the slide the cover slip should just slide off

2. Transfer the slide to dish or Coplin jar with 2X SSC/0.2% SDS at 50° C for 10 minutes

3. Transfer to dish or Coplin jar with 2X SSC for 15 minutes

4. Transfer to dish or Coplin jar with 0.2X SSC for 15 minutes

5. Transfer to 50 ml tube and spin at 500 rpm in clinical centrifuge for 5 minutes to dry

6. Add microarray slide to package, tape shut, send to Malcolm for scanning. Include your name and list of slides enclosed. Be sure to email Malcolm in advance (one week is nice) to be certain of his schedule for scanning.

7. Recieve electronic data and begin processing (see post-tif file protocol - coming soon).