Quantifying Gene Expression in Drosophila Clones

Total RNA from Eye-antenna imaginal discs containing wild-type or mutant clones was isolated using a Trizol RNA extraction method (Invitrogen). The SuperScriptIII First-Strand Synthesis System (Invitrogen) kit was used to synthesize complementary DNA. Real-time PCR was carried out on an Applied Biosystems machine using the SYBR green fast kit (Applied Biosystems) following the manufacturer's instructions. Relative gene expression was obtained from triplicate runs normalized to Rp49 as endogenous control. The following primers were used: Rp49, 5′-GGCCCAAGATCGTGAAGAAG-3′ and 5′-ATTTGTGCGACAGCTTAGCATATC-3′; Spitz, 5′-CGCCCAAGAATGAAAGAGAG-3′ and 5′-AGGTATGCTGCTGGTGGAAC-3′
Clones of mutant cells in the eye-antennal discs were generated using standard MARCM system56 (link). Immunostaining and Immunoprecipitation experiments were performed as previously described57 (link).