Amino-Allyl cDNA Labeling

互联网2013-09-06

1460

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 H2 O)
1 N NaOH, 0.5 M EDTA, 2 M HEPES
0.2 M NaHCO3 (bicarbonate), 4 M NH2 OH
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 H2 O
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 32 P-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 H2 O. 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 H2 O
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 NH2 OH
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 H2 O, 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 H2 O
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.