cbil.upenn.edu:RAD.Protocol:5895:Protocol - cbil.upenn.edu:RAD.Protocol:5895:Protocol

Amplified RNA was thawed and dried using a vacuum dryer at low heat setting. RNA was reconstituted in 9 ul coupling buffer (Amino Allyl MessageAmp aRNA kit, Ambion, Austin, TX). Mono-functional NHS ester Cy3 or Cy5 dye, reconstituted in 11 ul DMSO, was added (Amersham Cy Dye Post-labelling Reactive Pack, GE Healthcare, UK). In all experiments, reference aRNA and sample aRNA were labeled with Cy3 and Cy5 fluorescent dyes, respectively, and were then incubated in the dark for 30 minutes at room temperature. 4.5 ul 4 M Hydroxylamine (Ambion, Austin, TX) was added for 15 minutes to quench the dye coupling reaction. Dye-coupled RNA was purified into nuclease-free water using the aRNA filter cartridges (Ambion MessageAmp kit) in order to remove excess dye. Final volume of dye-conjugated RNA was 150 ul.
All experiments using protocol cbil.upenn.edu:RAD.Protocol:5895:Protocol: (E-CBIL-42, E-CBIL-43, E-CBIL-47)