Climbing third instar larvae were dissected in saline by tearing them immediately posterior to the mouth hooks using paired forceps and everting them. Males and females (as identified by the clear gonadal tissue embedded in fat body of the A5 segment) were dissected separately, and preparations were combined in equal numbers for RNA processing. The digestive system was pulled away from the body immediately anterior to the proventriculus.

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.