First strand synthesis of mRNA into eDNA was performed using First strand eDNA synthesis kit from Invitrogen following manufacturer's protocol. Briefly, 4 jlg of total RNA was denatured at 65°C for 5 min in the presence of Oligo dT 16 and dNTPs and incubated at 42°C for another 2 min with DTT, MgCb, and RNaseOUT in 10 X reverse transcription buffer. 1 IlL/reaction of the Superscript Reverse Transcriptase enzyme was added to the denatured RNA and incubated at 42°C for 50 min. The enzyme was denatured by heating at 70°C for 15 min. The reaction was completed by a quick high-speed centrifugation and the complementary RNA strand degraded by incubating with RNaseH for 20 min at 37°C. The preparation was stored at -70°C.

First strand synthesis by reverse transcription