P-CAGE-25560 - P-CAGE-25560

Aminoallyl labeling of cDNA for microarrays

To 5 μg aRNA, 2 μl of random hexamer primers (3 mg/ml) were added and the final volume was brought up to 18.5 μl with RNase-free water. After a 10 min incubation at 70ºC, the mix was transferred to ice for 1 min and centrifuged briefly. The remaining steps were done at room temperature. A 50 × aminoallyl-dNTP mix containing 25 mM dATP, 25 mM dCTP, 25 mM dGTP, 15 mM dTTP and 10 mM amino-allyl-dUTP (Sigma-Aldrich, St Louis, Missouri, USA) was prepared. To each sample, 6 μl of First Strand Buffer (Invitrogen), 3 μl 0.1 M DTT (Invitrogen) and 0.6 μl 50 × aminoallyl-dNTP mix and 2 μl SuperScript III reverse transcriptase (Invitrogen) was added. The resulting mix was incubated at 42ºC overnight to produce first-strand cDNA with aminoallyl-dUTP incorporated. RNA was hydrolyzed by addition of 10 μl of 1 M NaOH and 10 μl of 0.5 M EDTA and incubation at 65ºC for 15 min. To neutralize the pH, 10 μl of 1 M HCl was added to each sample.

To remove unincorporated aminoallyl-dUTP and free amines, the aminoallyl-labeled cDNA was purified. The cDNA reaction was mixed with 300 μl Buffer PB (Qiagen) and transferred to a QIAquick column (Qiagen). The column was centrifuged at 16060 × g for 1 min after which the eluate was re-added and centrifugation repeated. Two washes were carried out using 650 μl phosphate wash buffer (5 mM KPO4, pH 8.0, 80% ethanol) with 1 min centrifugation at 16060 × g following each wash. After drying the membrane by 1 min centrifugation at 16060 × g, purified cDNA was eluted twice in 30 μl phosphate elution buffer (4 mM KPO4, pH 8.5). The sample was dried to completion in a vacuum centrifuge, and the aminoallyl-labeled cDNA was resuspended in 4.5 μl 0.1 M Na2CO3, pH 9.0. To each sample, 4.5 μl of Cy5 dye ester (GE Healthcare) was added and the reaction was incubated for 1 hour in the dark at room temperature. A total of 35 μl 100 mM NaOAc, pH 5.2, and 250 μl Buffer PB (Qiagen) was added, the mix was transferred to a Qiaquick spin column (Qiagen) and centrifuged at 16060 × g for 1 min. The eluate was re-added and the centrifugation repeated. A single wash using 650 μl Buffer PE (Qiagen) with 1 min centrifugation at 16060 × g was carried out. After a 1 min additional centrifugation at 16060 × g to dry the membrane the labeled cDNA was eluted twice in 30 μl Buffer EB (Qiagen). The labeled cDNA was analyzed on a Nanodrop to assess dye incorporation.

Amount of nucleic acid labeled, Amplification, Used Label
All experiments using protocol P-CAGE-25560: (E-ATMX-22, E-ATMX-23)