Gene Expression Changes in Mated Flies

To quantify mating-induced changes in gene expression, the ovaries from 2 adult female flies (4-day-old virgin or mated females) were dissected. Total RNA was extracted using RNAiso Plus reagent (TaKaRa). cDNAs were prepared with ReverTra Ace qPCR RT Master Mix with gDNA remover (ToYoBo). qRT-PCR was performed using the Universal SYBR Select Master Mix (Applied Biosystems) with a Thermal Cycler Dice TP800 system (TaKaRa). Serial dilutions of a plasmid containing the ORF of each gene were used as a standard. The amount of target RNA was normalized to an endogenous control ribosomal protein 49 (rp49), and the relative fold change was calculated. The primers for quantifying nobo, nvd, sro, spo, phm, dib, sad and shd were used in the previous studies [64 (link)–66 (link)].