cbil.upenn.edu:RAD.Protocol:2346:Protocol - cbil.upenn.edu:RAD.Protocol:2346:Protocol
10 ug of total RNA was primed by 5 ug of a dT(16) oligomer and the mixture was heated to 70C for 10 min, and then transferred to ice. A premixed solution, consisting of 200 U Superscript II (Gibco), buffer, 100mM DTT 0.5mM dNTP, was added to the RNA. The reaction was then incubated at 42C for 2 hours. Nucleotides were used at these final concentrations: 0.5 mM for dATP, dCTP, and dGTP and 0.1 mM for dTTP and 0.4mM for aminoallyl-dUTP (Sigma ref: A0410). NaOH and EDTA were then added and RNA was hydrolyzed by incubation at 65C for 15min. After neutralization using Hepes, the aminoallyl cDNA was purified using a Bio-spin6 column (BioRad, ref: 732-6002) and concentrated using Centricon-30 microconcentrators (Amicon, ref 42410). NaHCO3 pH9 was added to a final concentration of 0.05M and each cDNA was combined with an aliquot of Cy3 or Cy5 monofunctional dye ester (Amersham, ref: PA25001/2) and incubated for an hour of coupling in the dark. Dye-coupled cDNA was purified using Qiaquick column (Qiagen ref: UB300) and concentrated with a Centricon-30 microconcentrator.