Quantitative Real-Time PCR Analysis of mRNA Expression

mRNA expression was assessed via quantitative real-time PCR (qRT-PCR)^{72 (link)}. Approximately 400 fly heads per n were isolated. Total RNA was isolated using TRIZOL and was reverse transcribed via oligo (dT) primers and Superscript II reverse transcriptase (Invitrogen; Carlsbad, CA, USA). qRT-PCR was performed using an Applied Biosystems Fast 7500 system with SYBR Green PCR master mix (Quanta Biosciences; Beverly, MA, UAS) and run in triplicate. Each qRT-PCR experiment was performed with independent RNA isolations and cDNA syntheses, and normalized to actin5C. Primers used (forward/reverse) were as follows: Cp1, 5′- CTCATGTGACGCTGCCCAAATC-3′/5′- CCAGCACAGGCGCCCTC-3′; GA25021, 5′- GACAGCATTGATTCTTCCCCTCC-3′/5′- GTGTGCCATTCCTCCTGGATG-3′; actin5C, 5′- AGCGCGGTTACTCTTTCACCAC-3′/5′- GTGGCCATCTCCTGCTCAAAGT-3′ (Fisher Scientific, Hampton, NH, USA). Primers for Cp1 readily detected endogenous Cp1 from repo-Gal4/+ flies, but this level of expression was not significantly altered in repo-Gal4/UAS-GA25021 flies (see Supplementary Fig. 5). Additionally, primers for GA25021 detected a product in repo-Gal4/UAS-GA25021 flies, but this signal was not altered by expression of Cp1 RNAi HMS00725 (see Supplementary Fig. 5). These findings confirm that the Cp1 and GA25021 primers were specific for their intended products.