Third instar larvae were cultured on food supplemented with 2.5% EtOH for 3 hours; 4.7% lethality was observed. Survivors were flash frozen in liquid nitrogen and stored at -80oC prior to RNA preparations.

Total RNA was isolated using Trizol, then Qiagen RNeasy spin columns. mRNA was isolated using Dynal oligo(dT) beads, then fragmented using divalent cations under elevated temperature, followed by sequential ligation of RNA linkers to the 5’ and 3’ ends. Next, reverse transcription was performed using a primer complementary to the 3’ linker and PCR was performed using primers complementary to both linkers. ~ 300 bp fragments were isolated from an agarose gel and gel-purified again.