Universal probe library assay design tool

RNA was extracted from the three anatomical regions dissected from 30 flies following the Trizol method. Reverse-transcription was made with qScript cDNA supermix (Quanta Biosciences). PCR primers were obtained using the Universal Probe Library Assay Design Tool (Roche, Basel, Switzerland): Opa1 forward, 5′-CATGGCACACTACTTTTCCTGA-3′, and reverse, 5′-TGCTACTAGCCGAGGAGCTAAT-3′, Marf forward 5′-CGCCAGTTGTTTGATGTTCA-3′, and reverse, 5′-ATTGGGCACACCACGAAT-3′, 4ebp forward 5′-CCAGATGCCCGAGGTGTA-3′, and reverse, 5′-AGCCCGCTCGTAGATAAGTTT-3′, InR forward 5′-CCGGGATTACTGTACTGAACCT-3′, and reverse, 5′-CGCCTGCTAAAGGATCTGA-3′. Expression levels were normalized by the expression of Rp49 with the following primer sequences: Rp49 forward, 5′- CGTTTACTGCGGCGAGAT-3′, and reverse, 5′-GCGCTCGACAATCTCCTT-3′. Quantitative real-time PCR was performed with SYBR Green from FastStart Essential DNA Green Master (Roche). Ct values were obtained using a real-time quantitative PCR system LightCycler 480 (Roche). Each sample was analysed in triplicate, and the expression was calculated according to the 2^−ΔΔCt method. Agarose gel electrophoresis was also performed with all the PCR products to check that the amplicon size was correct and to rule out non-specific amplification.