AminoAllyl cDNA Labeling

Materials

NotesThis 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 H2O2. 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 Hydrolysis1. To each sample add: 12.5 1N NaOH 5 µL 0.5 M EDTA2. Incubate at 65ºC, 15 min. 3. Neutralize each sample with: C. Cleanup on GFX columns1. 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: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 H2O2. To a Cy-dye aliquot add: 2.25 µL 0.2 M NaHCO33. Quickly combine dye and DNA.4. Incubate at r.t. in dark for 1 hr.5. Quench by adding 4.5 µL 4 M NH2OH6. Incubate at r.t. in dark for 15 min.E. Cleanup in QiaQuick columns1. Combine Cy5 and Cy3 samples (experiment and control).2. Add: 70 µL H2O, 500 µL Buffer PB3. 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 Solution1. To each sample/control combination add: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.