General Protocol for Poly A prep

These are the general protocols followed to make poly A RNA for genome array analysis of yeast using spotted arrays.

Where to get arrays:

For 100 ml cultures, yield will be 5-15 mg RNA.
Poly A+ is typically 2%

Try to assemble 10-15 ug poly A+ RNA for microarray experiments

Reverse Transcription labelling Protocol

a given reverse trancriptase reaction to make probe might
typically use 2 ug of once-selected polyA+ RNA, and yields enough
probe for two hybridization experiments.

Total RNA prep for Yeast. (from Joe DeRisi Pat Brown's Lab)

Culture Volume: 100mL

1. Resuspend pellet in 4mL of AE buffer. (50mM NaOAc, 10mM EDTA, pH 5.2)
2. Add 400uL of 10% SDS. Vortex.
3. Add 5mL of phenol equilibrated with AE buffer. (typical acid phenol prep)
4. Incubate at 65C, 10 minutes. Vortex.
5. Spin down debris. (10000rpm, in a Beckman JA-20)
6. Collect the aqueous phase.
7. Extract 2x with Chloroform
8. Add 1/10th volume 3M NaOAc, pH 5.2.
9. PPT with 2.5 volumes EtOH.
10. Wash 2x with 70% EtOH.
11. Resuspend in water.
12. Store at -80C.

I do the whole thing in falcon tubes, and oakridge tubes.
Run some stuff on a gel to verify you've got intact total RNA.

Poly A+ Prep

A single extraction with oligo-dT cellulose is good enough 
to use in the RT reaction to make probe for the micro arrays.

You can use either of the following protocols. I've used both
and they both work fine, although the oligo-dT spin coloumns
are easier, faster, and the return volume (though not the yield)
is greatly reduced.

mRNA purification kit Pharmacia 27-9258-01
(about 100 bucks for 4 columns - so the price actually
rivals straight oligo-dT resin)

NEB has an oligo-dT kit out which is a much better deal than
the Amerscam Pharmacia kit.

The spin column kits include instructions (of course)
but basically:

prepare columns by rinsing with equilibration buffer (40 minutes)
load RNA (1.25 mg)
rinse RNA
repeat 5x
elute with 65 C 5 mM Tris
elutes in approx. 1 ml
ppt or use directly
The whole prep takes about an hour.

Typical yield from this prep is 25 ug. The eluted RNA can be spin
 vacc'd to decrease the volume for a precipitation in 1 eppendorf tube
if you prefer.

Also, the prep will sometimes yield a tiny amount of resin fines that appear
as a small pellet upon spinning the resuspended RNA. These can be avoided by a brief spin of the RNA before pipetting it into your RT reaction, or by spinning and moving the sup to a new tube.

Conventional PolyA+ Selection:

For 2mg of Total RNA:

- Wash 200mg of oligo dT cellulose (collaborative research, cat. 20002)
with 10mL of NETS buffer (100mM NaCl, 10mM EDTA, 10mM Tris HCl pH 8.0, 0.2% SDS)
- Spin down, aspirate, repeat twice
- Resuspend in 1mL 2xNETS
- Add Total RNA in volume of 1mL
- Mix on a gentle shaker at RT for 1 hour
- Pour slurry onto a fritted funnel over a vacuum flask.
  You can also use disposable BioRad columns (10 ml plastic with frit)
  over a small vac flask using a rubber stopper 
  with a hole in it as a manifold
  with a 125 ml vac flask the spacing is just right so you can collect
  the eluates in 14 ml plastic falcon tubes placed inside the flask
- Wash resin with 1xNETS five times, 3mL each.
- Elute from funnel into a new vacuum flask (or use a new falcon tube)
  with 1mL, 65C prewarmed 1xETS
  buffer (Same as NETS, except no NaCl)
- Repeat 7 times
- Add 1/10th volum 3M NaAcetate, with linear acrylamide as carrier  (20ug).
- Add equal volume of isopropanol, chill to -20C, then precipitate.
- Wash pellet with 70% EtOH.
- Resuspend in water.
- Quantitate with a spectrophotometer

Linear Acrylamide (Carrier):

Unlike glycogen, linear acrylamide does not inhibit polymerase activities,
or restriction enzymes.

assemble in eppendorf tube:

5 mg Acrylamide
dissolve in 200 ul TE
add 1 ul 10% APS
add 1 ul TEMED
allow to polymerize (incubate as long as you like can heat to 37 to increase rate)

forms a jelly mass
add 2.5 volumes ethanol
spin 5-6 minutes
speed vac
resuspend 500 ul big water (ddH20) (i.e. 10 ug/ul)

takes a while to resuspend, but it does turn clear and resuspend
eventually (maybe leave it on your bench overnight). Subsequent
pellets are asy to resuspend.

use 10-20 ug for a precipitation
good for oligo size stuff and bigger.

resulting pellets are easy to resuspend.