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Amino-allyl cDNA labeling
M. Fero.  v.4
Amino-Allyl cDNA Labeling
Materials
RNA sample 30 µg Total or 2 µg mRNA
Oligo dT (dT18VN) 5 µg/µL, 34.8 (µg/mL)/A260
Superscript II, 5x 1st strand buffer (Invitrogen)
1 M DTT (Sigma, 43816, $16.80/10 mL)
50x dNTP Mix (dATP, dCTP, dGTP @25 mM, TTP=15mM)
10x AA-dUTP, 2 mM (MW = 523 g/mol), (Sigma A0410, $193.50/1mg - dissolve in 9.5 mL H2O)
1 N NaOH,  0.5 M EDTA,  2 M HEPES
0.2 M NaHCO3 (bicarbonate), 4 M NH2OH
GFX spin columns
QiaQuick columns
PolyA (10 mg/mL) - Sterile filtered (0.45 µm)
20x SSC (175.5 mg/mL NaCl, 88.3 mg/mL NaCitrate)
Cy3 and Cy5 Mono-reactive dye (Amersham PA23001 and PA25001): 
        To 1 tube dye add 36 µL anyhdrous DMSO and divide into 16x 2µL aliquots
       Anhydrous DMSO:  DMSO + Molecular Sieves (Sigma #M0133)

Notes
This procedure produces 1st strand cDNA with an anchored oligo dT primer and AA-UTP.  It then couples couples amino reactive Cy-dyes to the AA-UTP.  For spotted arrays control RNA should also be labeled using the opposing dye.

Procedure
A.  1st Strand Synthesis

1.  In a 1.5 mL tube add:
            RNA (30 µg total or 2 µg mRNA)
            1 µL (5 µg/µL) Oligo dT
            q.s. 25 µL H2O
2.  Incubate 70ºC, 10 min, then place on ice for 10 min.
3.  Meanwhile, prepare RT Master Mix (per sample):
            2   µL Superscript II
            8   µL 5x 1st strand buffer
            0.4 µL 1 M DTT
            0.8 µL 50x dNTP mix
            4  µL 10x  AA-dUTP  (optional: spike with 1 µL 32P-dCTP)
5.  Add 15 µL of RT Master Mix to each sample.
4.  Incubate 42ºC, 2 hrs.

B.  RNA Hydrolysis
1.  To each sample add:
            12.5   1N NaOH
            5 µL 0.5 M EDTA
2.  Incubate at 65ºC, 15 min.
3.  Neutralize each sample with:
20 µL 2M HEPES
O.K. to store o.n. at 4ºC.

C.  Cleanup on GFX columns
1.  Turn on Speed Vac refrigerators.
2.  Add 0.5 mL GFX capture buffer.  (optional: save 1 uL for 32P counts)
3.  Load on GFX column.  Spin 1 min. Discard flow-through.
4.  Add 0.5 mL GFX wash buffer.  Spin.  Discard flow-through.
5.  Elute in amber 1.5 mL tube:
Add 50 µL H2O.  Incubate 1 min.  Spin 1 min.
Repeat elution a 2nd time into the same tube.   (Optional: save aliquot to measure 32P counts)
7.  Dry in speed-vac.

D.  Coupling to amino-reactive Cy dyes  (e.g. Cy5 for sample, Cy3 for control)
1.  Resuspend in 4.5 µL H2O
2.  To a Cy-dye aliquot add: 2.25 µL 0.2 M NaHCO3
3.  Quickly combine dye and DNA.
4.  Incubate at r.t. in dark for 1 hr.
5.  Quench by adding 4.5 µL 4 M NH2OH
6.  Incubate at r.t. in dark for 15 min.

E.  Cleanup in QiaQuick columns

1.  Combine Cy5 and Cy3 samples (experiment and control).
2.  Add:  70 µL H2O, 500 µL Buffer PB
3.  Apply to a QiaQuick column.  Spin 13K g for 30".  Discard flow-through.
4.  Add 750 µL Buffer PE.  Spin.  Discard flow through.
5.  Repeat wash with PE.
6.  Spin 1 min. to dry column.
7.  Elute:  Add 30 µL buffer EB, incubate 1 min.  Spin 1 min. into a fresh amber tube.
8.  Repeat elution 1x.
9.  Speed-vac to dry down.

F.  Preparation of Hybridization Solution
1.  To each sample/control combination add:
27.8 µL H2O
5.4 µL 20x SSC
2.8 µL polyA  (10 mg/mL)

2.    Load the DNA mixture on a 0.5 µm Millipore spin column
       Spin at 12,000 g x5 min.

3.  Store at -20ºC in the dark. 

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