Isolating RNA from the samples to be compared
Converting the RNA samples to labeled cDNA via reverse transcription; this step may be combined with aRNA amplification
Hybridizing the labeled cDNA to identical membrane or glass slide arrays
Removing the unhybridized cDNA
Detecting and quantitating the hybridized cDNA, and
Comparing the quantitative data from the various samples
**This doesn’t include array fabrication
I. RNA Isolation
1. Aspirate media from cells and wash once with Phosphate Buffered Saline (PBS).
2. Add 5mL Trypsin (1X) and incubate at 37°C for approximately 5 minutes.
3. Visually inspect plate to ensure cells have detached.
4. Add 4mL of cell media to plate and pipet cell suspension into a 50mL polypropylene conical-bottom tube.
5. Wash plate with an additional 4mL of media and add it to the tube.
6. Pellet the cells by centrifugation at 1000 rmp for 6 minutes at 4 °C and discard supernatant.
7. Add 2mL Trizol (Life Technologies; Cat # 15596-014) per ~2X106 cells to the pellet and pass the suspension through an 18 gauge syringe several times to disrupt the pellet.
8. Incubate the sample at room temperature for 5 minutes.
9. Add 0.2mL chloroform per 1mL Trizol and shake vigorously for 1 minute.
10. Incubate at room temperature for 2.5 minutes.
11. Remove cellular debris by centrifugation at 4000 rpm for 15 minutes at 4 °C.
12. Transfer supernatant to 1.2mL microcentrifuge tubes (0.5mL/tube) and an equal volume of isopropanol to precipitate the RNA.
13. Incubate at room temperature for 15 minutes.
14. Centrifuge at 15,000 rpm for 15 minutes to pellet RNA.
15. Discard supernatant and resuspend the pellet in 70% ethanol. RNA can be stored in 70% ethanol at -20°C until use.
16. Prior to use, centrifuge at 15,000 rpm for 15 minutes and discard supernatant.
17. Resuspend pellet in diethylpyrocarbonate (DEPC) treated water or Rnase-free TE buffer for labeling.
II. cDNA Synthesis and Labeling
1. Prepare a labeling reaction master mix containing 500mM dCTP, 500mM dATP, 500mM dGTP, 100mM dTTP, 1mM dithiothreitol (DTT) and 1X RT buffer.
2. To 10mg of total RNA in a microcentrifuge tube, add 2mg of oligo(dT) and DEPC-treated water to a total volume of 10mL.
3. Incubate the reaction mixture at 70°C for 10 minutes and chill on ice for one minute.
4. Add 15mL of RT labeling mix, 3mL cyanine3-dUTP or cyanine5-dUTP (1mM), 2mL Superscript II RT (200U/mL; Life Technologies; Cat# 18064-014).
5. Mix thoroughly and incubate at 42°C for 2 hours.
6. Briefly centrifuge the reaction and add 1.5mL of 20mM EDTA to stop the reaction.
7. Add 1.5mL of 500mM NaOH and heat at 70°C for 10 minutes to degrade the RNA.
8. Neutralize reaction by adding 1.5mL of 500mM HCl.
9. Unincorporated fluorescent nucleotides are removed by glass fiber filtration using GFX columns (Pharmacia Cat# 27-9602-01) and the instructions provided by the manufacturer.
10. Elute purified products using 50mL of TE pH 8.0 and dry the probe to completion.
11. Resuspend the probe in 10mL of DEPC treated water.
12. Repeat steps 2-11 for other probe.
III. Prehybridization
1. Prepare prehybridization buffer containing 5X SSC, 0.1% SDS, and 1% bovine serum albumin (BSA)
2. Prepare 2X hybridization buffer containing 50% formamide, 10X SSC, and 0.2% SDS.
3. Place slides to be analyzed into a Coplin jar, fill with prehybridization buffer, and incubate at 42°C for 45 minutes.
4. Wash the slides by dipping 5 times in room temperature MilliQ water.
5. Dip slides in room temperature isopropanol and air dry.
*Slides should be used immediately following prehybridization.
IV. Hybridization
1. Combine 10mL each of purified Cy3- and Cy5-labeled probes, mix well and add
1mL COT1-DNA (20mg/mL; Life Technologies; Cat# 25279-011) and 1mL Poly(A)-DNA (20mg/mL; Pharmacia; Cat# 27-7836-01) to block nonspecific hybridization.
2. Heat the probe mixture at 95°C for 3 minutes to denature.
3. Centrifuge the probe at maximum angular velocity for 1 minute.
4. Combine the probe with an equal volume of 2X hybridization buffer that has been heated to 42°C.
5. Apply the labeled probe to a prehybridized microarray slide and cover with a 22mm X 60mm polyethylene hydrophobic coverslip.
6. Place the slide in a sealed hybridization chamber, add 20mL of water to the chamber at the end of the slide.
7. Place the sealed chamber in a 42°C water bath and incubate for 16-20 hours.
8. Remove the array from the hybridization chamber, taking care not to disturb the coverslip.
9. Place the slide in a staining dish containing low-stringency wash buffer containing 1X SSC and 0.2% SDS at 42°C.
10. Gently remove the coverslip while the slide is in solution and agitate for 4 minutes.
11. Wash the slide at high stringency in a staining dish containing 0.1X SSC and 0.2% SDS at room temperature, agitating for 4 minutes.
12. Wash the slide in 0.1X SSC, agitating for 4 minutes.
13. Allow slides to air dry.