Total RNA preparation

1. Spin down yeast quickly (3500g for 3-5 minutes) at room temperature.  Remove supernatant.
2. Resuspend in 7 ml lysis buffer (10mM Tris 7.5, 10mM EDTA, 0.5% SDS) per 100 ml per OD culture.
3. Add 7 ml acid phenol per 100 ml per 1 OD culture.  Vortex well.  Transfer to 50 ml Oak Ridge tubes.
4. Incubate at 65° for 30 minutes with occasional vortexing.
5. Place on ice 5 minutes.  Centrifuge at 4° at 10,000 rpm (in JA-20 rotor) for 10 minutes.
6. Remove aqueous (upper) layer.  Add equal volume phenol.  Mix.  Spin as before.
7. Add equal volume chloroform.  Mix.  Spin as before.
8. Add sodium acetate to 0.3M.  Add 2 volumes 100% ethanol.  Cool at -20° for 30 min. Spin as before.  Wash twice with 70% ethanol.
9. Dry 1-2 hours at room temperature until all ethanol is gone.  Do NOT overdry.
10. Resuspend in 0.5 ml 10mM Tris 7.4 per 100 ml of culture.
11. Measure yield by OD 260-280 nm (RNA is 40 ug/ml/A260; A260/280 = 2).

Isolation of poly(A) mRNA from total RNA using a Qiagen Oligotex mRNA kit

1. Dilute 3 mg RNA to 650 ul.  Add 650 ul Buffer OBB.  Add 200 ul Oligotex.
2. Mix gently.  Incubate for 3 minutes at 70°C.
3. Hybridize at room temperature for 60 minutes.
4. Centrifuge for 2 minutes and remove most of supernatant.
5. Resuspend in 600 ul Buffer OW2.  Vortex.  Pipet onto a Maxi column.  Centrifuge at maximum speed 1 minute.
6. Repeat with 600 ul Buffer OW2.
7. Transfer column to clean tube.  Add 100 ul 70°C Buffer OEB.  Resuspend resin.  Incubate resin briefly at 70°C.  Centrifuge 1 minute.

Length fractionation and RNA recovery

1. Prepare a 1X TAE, 1.2% LMP (low melting point) agarose gel.
A. TAE buffer is the best for both RNA gels and for nucleic acid recovery.  It only has sufficient buffering capacity during extended runs if there’s recirculation however.  I used an Owl model A5 Buffer Puffer™ horizontal gel electrophoresis system.  Its "passive" recirculation is sufficient.
B. Ideally a LMP agarose gel less than 1% would provide the best separation for RNAs 1.5 kb and up.  In my experience, at 1% a LMP agarose gel is too fragile to survive virtually any manipulation.
C. Do not use any kind of denaturant in the gel or the running buffer.  They all smear the RNA at least a little bit.  Especially do not use formaldehyde.  It works fine for Northern blots, but it does something irreversible to the RNA that prevents reverse transcription.
2. Equilibrate gel and running buffer at 4°C.
A. The gel is run at 4°C to prevent overheating which can smear bands and melt the gel.  I have my electrophoresis apparatus set up in a 4°C cold room.
3. Heat denature RNA sample and RNA ladder in formamide and 10mM EDTA at 65°C for 10 minutes.  Place on ice, add loading dye, and load onto gel immediately.
A. For a ladder I used Ambion’s Millenium™ and Century™ RNA markers.  I added 0.5 ul of each ladder (1 ug/ul) and 0.2 ul of 0.5 M EDTA to 8 ul molecular biology grade formamide.
B. For my yeast sample I added 50 ul poly(A) RNA at 1ug/ul and 3 ul of 0.5 M EDTA to 100 ul formamide.
C. I added 1 ul loading dye (0.25% Bromophenol Blue, 0.25% Xylene Cyanol, 20% Ficoll) to the ladder and 15 ul to the yeast sample.
D. I find that RNA can overload an agarose gel if the lane isn’t wide enough.  I recommend well width of at least 1 mm per microgram of sample RNA.  For the 50 ug of RNA I loaded, I taped six teeth together to produce a 5 cm wide lane in the middle of the gel.
4. Run at 9 V/cm until the desired separation length is reached.
A. I ran at 260 V for 2 hours.  The bromophenol blue migrated about 10 cm.
5. Excise ladder lane.  Stain with ethidium bromide, and visualize with a UV transilluminator.
A. The purpose of this step is to decide where to cut your length fractions.  I started at 300 bps and worked my way up.
6. Excise your desired length fractions from the gel.
A. In order to do this, I taped a sheet of metric graph paper to my bench, and secured the gel (still in the gel tray) over it.  Using the graph paper lines as a guide, I cut thirty 2 mm slices with a scalpel.  I had originally designed a cookie cutter-like device to precisely cut several slices at once.  At 2 mm, however, the device tended to mash the gel preferentially to cutting it.  So unless you’re cutting larger slices or you have a real cool robot arm to work with, I’d just cut the gel by hand.
7. Melt each slice at 70°C for 10 minutes.
8. Transfer to 42°C.  Add 1 U of β-agarase per 200 ul of gel.  Add exogenous doping control RNAs.  Vortex.  Incubate at 42°C for 2 hours.
A. I used agarACE from Promega.  It probably doesn’t matter where you get it from, but its activity goes down over time, so use fresh enzyme.
B. The doping control RNA is used to normalize the various microarrays since each length fraction will have a different RNA recovery yield and a different number of enriched genes.
9. Concentrate each agarase reaction in a Microcon-30 spin column.  Wash twice with water and elute.
A. I tried a number of methods to recover the RNA from the agarase reaction.  The Microcon-30 method had by far the best yield for every length of RNA.
B. The Microcon-30 columns do trap the β-agarase and some undigested agarose.  I find that neither of these have any effect on reverse transcription, but they do effect UV absorbance in case you were interested in measuring RNA concentration by OD.
C. After eluting, I diluted each sample to 30 ul and used 10 ul per array.  I recommend length separating enough RNA to do duplicates or triplicates since not every microarray hybridization goes perfectly.

RNA labeling

1. RT reaction
ul
Oligo dT primer (10 ug/ul) 0.5
dN9 primer (10 ug/ul) 1.0
RNA 10
Water 6.9

Incubate at 70ºC for 10 minutes.  Chill on ice.

ul
5X Buffer 6.0
DTT (0.1 M) 3.0
50X dNTP mix* 0.6
Superscript II 2.0

* 50X dNTP mix 1X dNTPs
dATP 25 mM 500 uM
dCTP 25 mM 500 uM
dGTP 25 mM 500 uM
dTTP 12.5 mM 250 uM
aa-dUTP** 12.5 mM 250 uM
**Sigma-Aldrich Catalog A-0410

Incubate at 42ºC for 90 minutes.

2. Hydrolysis
Add:
10 ul 0.5M EDTA
10 ul 1N NaOH

Incubate at 65°C for 10 minutes.
Neutralize with 25ul 1M HEPES pH 7.

3. Cleanup
Fill one Microcon-30 concentrator with 425 ul water.
Add neutralized reaction.  Spin.  Wash twice with water.  Elute cDNA in less than 10 ul.

4. Aliquot dye
Resuspend the contents of one pack of the monofunctional NHS-ester Cy3 or Cy5 dye (mono-functional Cy3 or Cy5 reactive pack, Amersham catalog PA 23001 and PA 25001) in 10 ul DMSO.  Make ten 1 ul aliquots.

5. Coupling
Add 1.5 ul 1M NaHCO3 buffer pH 9.0 to cDNA for a final NaHCO3 concentration > 0.1 M.  Add to a 1 ul aliquot of Cy3 or Cy5 dye.  Mix dye and cDNA vigorously. Let incubate 1 hour at room temperature in the dark.  Immediately dry unused aliquots in a speed vac without heat.  Store dried aliquots at 4ºC in a vacuum dessicator.  Never although the dye to come in contact with water before coupling.  cDNA can be added directly to the dried aliquots in future coupling reactions.

6. Cleanup
To remove unincorporated Cy dyes use Qia-quick PCR purification kit.
Add 150 ul Buffer PB.
Apply to Qia-quick column and spin at 13K for 1 minute.
Wash twice with 750ul Buffer PE.  Spin for 1-2 minutes at high speed to dry column.
Elute with 50ul Buffer EB.
Combine Cy5 and Cy3 samples.  Dilute to 500 ul with water and Microcon-30 concentrate.  Elute in less than 20 ul.

DNA microarray hybridization

1. Hybridization solution preparation
A 30 ul hybridization volume is best for a standard 32-tip array and a glass coverslip.

ul
Probe 20.8
20X SSC 6.0
polyA RNA (10ug/ul) 2.0
1M HEPES pH 7.0 0.6
10% SDS 0.6
30

2. Hybridization
Place a microarray in a hybridization chamber.  Add water or 3X SSC to the chamber for hydration during the hybridization.
Denature probe by incubating hybridization solution at 100º for 1.5-2 minutes.  Briefly vortex and centrifuge the hybridization solution.  Pipet the solution onto the center of the array.  Carefully place a glass coverslip over the spotted area of the microarray.  Quickly close the hybridization chamber and place in a 65º water bath.  Prepare no more than two microarrays at a time.
Allow hybridization to proceed for 18 hours.

3. Prepare three wash solutions

Wash1 Wash2 Wash3
20X SSC 15 ml 3 ml 0.75 ml
10% SDS 1 ml 0 ml 0 ml
Water 284 ml 297 ml 299.25 ml

Place a slide rack in wash solutions 1 and 2.
Heat wash solution 1 to 65º before use.

4. Washing microarrays
Remove a hybridization chamber from the 65º water bath.  Open the chamber and quickly place the microarray into wash solution 1.  Let it rest on top of the slide rack facing down.  That way when the coverslip comes loose it floats down, straight away from the array, and is less likely to cause scratches.  When the coverslip has fallen away place the slide on its side in the rack as usual.  Repeat with other microarrays until all are in wash solution 1.  Incubate slides and wash solution at 65º for 15 minutes.
Manually transfer each slide to the new rack in wash solution 2.  Wash the slides for 10 minutes shaking gently on a orbital shaker.
Transfer rack and slides to wash solution 3 and wash for 10 minutes shaking gently on a orbital shaker.
Spin at 600 rpm for 1 minute to dry.