P-GSE40140-14 - P-GSE40140-14

labelling protocol
10 µg of total RNA were primed with 1.25 µl of 100 µM Oligo dTV DNA primer at 70°C for 10 min, then reversed transcribed at 42°C for 1 h in the presence of 400 U SuperScript II RTase (Invitrogen), and 500 µM each dATP, dCTP, dGTP, with 100 µM dTTP, 400 µM aminoallyl-dUTP. The aminoallyl-labeled cDNA was hydrolyzed to obtain single stranded labeled DNA with addition of 10 µl of 0.5 M EDTA and 10 µl of 1 N NaOH at 65℃ for 15 min followed by neutralization with 25 µl of 1 M HEPES, pH 7.4. The labed DNA was then purified with Microcon-30 concentrator by washing with water for three times. NHS-ester Cy dyes were used to couple with the aminoally-cDNA at room temperature for 1 h. Unreacted NHS-ester Cy dyes were quenched with addition of 4.5 µl of 4 M hydroxylamine and removed by PCR clean up kit (QIAGEN). For details, see
Experiment E-GEOD-40140